Programming self-organizing multicellular structures with synthetic cell-cell signaling

Abstract

A common theme in the self-organization of multicellular tissues is the use of cell-cell signaling networks to induce morphological changes. We used the modular synNotch juxtacrine signaling platform to engineer artificial genetic programs in which specific cell-cell contacts induced changes in cadherin cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems: robust self-organization into multidomain structures, well-choreographed sequential assembly, cell type divergence, symmetry breaking, and the capacity for regeneration upon injury. The ability of these networks to drive complex structure formation illustrates the power of interlinking cell signaling with cell sorting: Signal-induced spatial reorganization alters the local signals received by each cell, resulting in iterative cycles of cell fate branching. These results provide insights into the evolution of multicellularity and demonstrate the potential to engineer customized self-organizing tissues or materials.

Fig. 1. Engineering cell-cell communication networks to program synthetic morphogenesis.

(A) Design logic underlying our synthetic morphogenesis circuits. Engineered cell-cell signaling is used to drive changes in cell adhesion, differentiation, and production of new cell-cell signals. These outputs can subsequently be propagated to generate new cell-cell signaling relationships. (B) Molecular components used for assembly of simple morphological circuits. We used two synNotch ligand-receptor pairs (surface ligands CD19 and GFP) for cell signaling, three fluorescent proteins as markers of “differentiation, “ and several cadherin molecules (expressed at different levels) as morphological outputs. Engineered circuits are transduced into L929 fibroblast cells, placed in defined numbers in low-adhesion U-bottom wells, and screened by microscopy for spatial self-organization.

Fig. 2. Engineering self-organizing multilayered spheroids.

(A to C) Two-layer circuit. (A) An A-type sender cell expressing CD19 ligand induces a B-type receiver cell to express E-cadherin and GFP. (B) SynNotch cell-cell signals drive receiver cells to express E-cadherin (Ecad), which leads to their segregation into a central layer. The system starts with two disordered cell genotypes but organizes to form a structure with two distinct spatial compartments. (C) Images of the spheroid at 1 and 24 hours. See fig. S1 for other data. (D to G) Three-layer circuit. (D) An A-type cell can send signals to a B-type cell using CD19 ligand, which induces expression of E-cadherin (high expression) and GFP_lig (surface-expressed GFP). The induced B-type cell can then send reciprocal signals to the A-type cell; GFP_lig serves as ligand to stimulate anti-GFP synNotch receptor expressed in the A-type cell. This reciprocal interaction is programmed to drive a low level of E-cadherin and mCherry. (E) Cell fate diagram showing how this program drives a two-step differentiation process in which the A→B synNotch signal first drives conversion of B-type cells to C-type cells that self-adhere and sort to the center of the structure. The sorted C-type cells then present the C→A synNotch signal (driven by GFP_lig) to convert spatially adjacent A-type cells into the middle-layer D-type cell (mCherry and low-level E-cadherin expression). A-type cells bifurcate into two phenotypes, depending on their spatial proximity to the C-type cells in the core of the structure. Here, the system starts with two disordered cell genotypes but self-organizes into three distinct cell phenotypes organized into three spatially distinct compartments. (F) Images from the development of the three-layer system from 0 to 20 hours. See fig. S2 and movie S1 for other data and time-lapse videos. (G) Formation of the three-layer structure is disrupted if synNotch signaling is inhibited (using DAPT, a g-secretase inhibitor) or if cadherins are not driven as outputs. See fig. S3 for more information.

Fig. 3. Three-layer self-organized structure is robust, reversible, and self-repairing.

(A) Distribution of structures generated in 28 independent wells (starting with 200 A-type cells and 40 B-type cells). About 90% of the wells showed formation of three-layer structures; the majority of these showed one spheroid per well, with the remainder showing either twinned spheroids or multiple independent three-layer spheroids. Example images of these structural subtypes are shown at the right. (B) Three-dimensional confocal reconstruction of a three-layer structure cross section, shown from two views. See movie S1 for full rotational view of the 3D structure. (C) Self-repair of a cleaved three-layer structure. The preformed spheroid was cleaved using a microfluidic guillotine, and the two resulting fragments were observed for 25 hours. The frames at 0 hours show the two fragments, with a dotted line indicating the cleavage plane that exposes the internal core of the spheroid. Images at 25 hours show self-repair of the spherical three-layer structure. (D) The structure is reversible if treated with the synNotch inhibitor DAPT. Within 3 days, the differentiation and spatial organization of cells disappeared. Original A- and B-type cells became randomly organized.

Fig. 4. Single-genotype circuit that induces fate bifurcation and spatial ordering into a two-layer structure.

(A) Design of single-genotype circuit with lateral inhibition between sender (CD19⁺) and receiver (antiCD19^- synNotch-activated) states. The cell encodes both CD19 and anti-CD19 synNotch, but activated synNotch receptor drives expression of tet repressor (tTS), which inhibits CD19 expression. Thus, neighboring cells will drive each other into opposite states indicated by red and green fluorescent markers (fate RED and GREEN). (B) E-cadherin expression driven from the synNotch-activated promoter. An initially homogeneous population of red cells undergoes bifurcation into RED fate and Ecad-positive GREEN fate by lateral inhibition, and GREEN-fate cells are finally sorted inside to form an inner core. The system starts with a single-genotype population but is expected to organize into a two-layer structure. (C) Purification of a homogeneous population by sorting for mCherry^hlgh/GFP^low cells. When allowed to communicate through lateral inhibition, the cells rebifurcate into two distinct fluorescently labelled populations (bottom). See fig. S4 and supplementary materials for more information on how the lateral inhibition circuit was constructed and executed. (D) Development of the single-genotype two-layer structure. Time frames are shown at 1, 25, and 50 hours, showing initial cell fate bifurcation followed by formation of a stable two-layer structure. See fig. S5 for more information and movie S3 for time-lapse video.

Fig. 5. Programming spherically asymmetric structures by inducing differentially sorting adhesion molecules.

(A) Logic of deploying alternative adhesion outputs to generate different spatial structures. In the spherically symmetric structures of Figs. 2 to 4, we used high and low levels of Ecad expression to define different populations of cells. High- and low-Ecad populations lead to sorting into concentric shells, because Ecad^lo cells still prefer to bind Ecad^hi cells. In contrast, two cell populations that express either Ncad or Pcad will sort into distinct compartments (nonconcentric) because each of these cadherins prefers homotypic self-association to heterotypic cross-association. (B) Three-layer asymmetric circuit I, with the same architecture as that shown in Fig. 2, except that B-type cells are induced to express Ncad and A-type cells are induced to express Pcad. In phase II of the development (reciprocal B→A signaling), the A-type cells become red and self-sort to form one to three external poles (with unactivated A-type cells associated at their periphery). The starting population included 100 cells of each type. When we started with only 30 cells of each type (right image), we reproducibly generated single-pole structures. See fig. S7 and movie S4 for more information, time-lapse videos, and 3D structure. (C) Three-layer asymmetric circuit II. An A-type cell constitutively expresses Pcad and mCherry as well as CD19 ligand. B-type cells recognize CD19 with anti-CD19 synNotch receptor, which drives expression of Ncad and GFP_lig. In reciprocal signaling, GFP_lig drives induction of a BFP marker in A-type cells. Here, the red A-type cells first form a central core and the induced green B-type cells form polar protrusions. A third cell type (blue) forms at the boundary between the red core and the green protrusions. See fig. S8 and movie S5 for more information, time-lapse video, and 3D structure. Information on other structures using different cadherin pairs is shown in figs. S9 and S10 and movies S6 and S7.

Fig. 6. Gallery of different self-organizing multicellular structures that can be programmed using the simple synNotch→adhesion toolkit.

(A) Gallery of spatially organized behaviors generated in this work, organized by resulting number of cell types and spatially distinct compartments as well as by increasing asymmetry. See table S1 for details of the construction of these 12 structures. Diagrams of several of the different three-layer structures are shown schematically below. (B) These synthetic developmental systems share the common principles in which cascades of cell-cell signaling, linked by morphological responses, lead to increasing diversification of cell types. As signaling drives morphological changes and reorganization, new cell-cell interactions arise, resulting in increasingly distinct positional information encountered by each cell in the structure.