Peeking under the hood of early embryogenesis: Using tools and synthetic biology to understand native control systems and sculpt tissues.

Early embryogenesis requires rapid division of pluripotent blastomeres, regulated genome activation, precise spatiotemporal signaling to pattern cell fate, and morphogenesis to shape primitive tissue architectures. The complexity of this process has inspired researchers to move beyond simple genetic perturbation into engineered devices and synthetic biology tools to permit temporal and spatial manipulation of the control systems guiding development. By precise alteration of embryo organization, it is now possible to advance beyond basic analytical strategies and directly test the sufficiency of models for developmental regulation. Separately, advances in micropatterning and embryoid culture have facilitated the bottom-up construction of complex embryo tissues allowing ex vivo systems to recapitulate even later stages of development. Embryos fertilized and grown ex vivo offer an excellent opportunity to exogenously perturb fundamental pathways governing embryogenesis. Here we review the technologies developed to thermally modulate the embryo cell cycle, and optically regulate morphogen and signaling pathways in space and time, specifically in the blastula embryo. Additionally, we highlight recent advances in cell patterning in two and three dimensions that have helped reveal the self-organizing properties and gene regulatory networks guiding early embryo organization.

Porous Transport Layers with Laser Micropatterning for Enhanced Mass Transport in PEM Water Electrolyzers.

Efficient electrochemical energy conversion technologies, such as fuel cells and water electrolyzers, require high current densities to lower the capital cost for large-scale commercialization but are often limited by mass transport. In this study, we demonstrated exceptional electrochemical performances in proton electrolyte membrane water electrolyzers (PEMWEs) creating micropatterned pore channels in the porous transport layer (MPC PTL) using a picosecond laser. This approach yielded an impressive performance of 1.82 V @ 2 A·cm-2, which is better than commercial PTL of 1.90 V @ 2 A cm-2. The significant performance enhancement is attributed to the micropatterned porous channel structure, facilitating the efficient expulsion of oxygen bubbles and input of reactant water. This work provides valuable insights for the design of PTL responsible for biphasic transport in electrochemical energy conversion technologies.

Controlling Physical and Biochemical Parameters of Actin Nucleation Using a Patterned Model Lipid Membrane.

Self-assembly of nanoscale actin cytoskeletal proteins into filamentous networks requires organizing actin nucleation areas on the plasma membrane through recruiting actin nucleators and nucleation-promoting factors (NPFs) to the areas. To investigate impacts of the nucleation geometry on actin network assembly, we localized NPF or nucleator on defined micropatterns of laterally mobile lipid bilayers confined in a framework of a polymerized lipid bilayer. We demonstrated that actin network assembly in purified protein mixtures was confined on NPF- or nucleator-localized fluid bilayers. By controlling the shape and size of nucleation areas as well as the density and types of localized NPFs and nucleators, we showed that these parameters regulate actin network architectures. Actin network assembly in Xenopus egg extracts was also spatially controlled by patterning bilayers containing phosphatidylinositol 4,5-bisphoshate (PI(4,5)P2), an essential lipid signaling mediator. Therefore, the system provides a promising platform to investigate the physical and biochemical principles for actin network assembly.

Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

Nanoparticle Assembly: From Self-Organization to Controlled Micropatterning for Enhanced Functionalities.

Nanoparticles form long-range micropatterns via self-assembly or directed self-assembly with superior mechanical, electrical, optical, magnetic, chemical, and other functional properties for broad applications, such as structural supports, thermal exchangers, optoelectronics, microelectronics, and robotics. The precisely defined particle assembly at the nanoscale with simultaneously scalable patterning at the microscale is indispensable for enabling functionality and improving the performance of devices. This article provides a comprehensive review of nanoparticle assembly formed primarily via the balance of forces at the nanoscale (e.g., van der Waals, colloidal, capillary, convection, and chemical forces) and nanoparticle-template interactions (e.g., physical confinement, chemical functionalization, additive layer-upon-layer). The review commences with a general overview of nanoparticle self-assembly, with the state-of-the-art literature review and motivation. It subsequently reviews the recent progress in nanoparticle assembly without the presence of surface templates. Manufacturing techniques for surface template fabrication and their influence on nanoparticle assembly efficiency and effectiveness are then explored. The primary focus is the spatial organization and orientational preference of nanoparticles on non-templated and pre-templated surfaces in a controlled manner. Moreover, the article discusses broad applications of micropatterned surfaces, encompassing various fields. Finally, the review concludes with a summary of manufacturing methods, their limitations, and future trends in nanoparticle assembly.

2D and 3D Micropatterning of Mussel-Inspired Functional Materials by Direct Laser Writing.

This work addresses the critical need for multifunctional materials and substrate-independent high-precision surface modification techniques that are essential for advancing microdevices and sensing elements. To overcome existing limitations, the versatility of mussel-inspired materials (MIMs) is combined with state-of-the-art multiphoton direct laser writing (DLW) microfabrication. In this way, 2D and 3D MIM microstructures of complex designs are demonstrated with sub-micron to micron resolution and extensive post-functionalization capabilities. This study includes polydopamine (PDA), mussel-inspired linear, and dendritic polyglycerols (MI-lPG and MI-dPG), allowing their direct microstructure on the substrate of choice with the option to tailor the patterned topography and morphology in a controllable manner. The functionality potential of MIMs is demonstrated by successfully immobilizing and detecting single-stranded DNA on MIM micropattern and nanoarray surfaces. In addition, easy modification of MIM microstructure with silver nanoparticles without the need of any reducing agent is shown. The methodology developed here enables the integration of MIMs in advanced applications where precise surface functionalization is essential.

[Spatial Constraints of Rectangular Hydrogel Microgrooves Regulate the Morphology and Arrangement of Human Umbilical Vein Endothelial Cells]. / çŸ©å½¢æ°´å‡èƒ¶å¾®å‡¹æ§½çš„ç©ºé—´çº¦æŸè°ƒæŽ§äººè„é™è„‰å† çš®ç»†èƒžçš„å½¢æ€å’ŒæŽ’åˆ—.

Objective: To construct microscale rectangular hydrogel grooves and to investigate the morphology and alignment of human umbilical vein endothelial cells (HUVECs) under spatial constraints. Vascular endothelial cell morphology and alignment are important factors in vascular development and the maintenance of homeostasis. Methods: A 4-arm polyethylene glycol-acrylate (PEG-acrylate) hydrogel was used to fabricate rectangular microgrooves of the widths of 60 µm, 100 µm, and 140 µm. The sizes and the fibronectin (FN) adhesion of these hydrogel microgrooves were measured. HUVECs were seeded onto the FN-coated microgrooves, while the flat surface without micropatterns was used as the control. After 48 hours of incubation, the morphology and orientation of the cells were examined. The cytoskeleton was labelled with phalloidine and the orientation of the cytoskeleton in the hydrogel microgrooves was observed by laser confocal microscopy. Results: The hydrogel microgrooves constructed exhibited uniform and well-defined morphology, a complete structure, and clear edges, with the width deviation being less than 3.5%. The depth differences between the hydrogel microgrooves of different widths were small and the FN adhesion is uniform, providing a micro-patterned growth interface for cells. In the control group, the cells were arranged haphazardly in random orientations and the cell orientation angle was (46.9±1.8)°. In contrast, the cell orientation angle in the hydrogel microgrooves was significantly reduced (P<0.001). However, the cell orientation angles increased with the increase in hydrogel microgroove width. For the 60 µm, 100 µm, and 140 µm hydrogel microgrooves, the cell orientation angles were (16.4±2.8)°, (24.5±3.2)°, and (30.3±3.5)°, respectively. Compared to that of the control group (35.7%), the number of cells with orientation angles <30° increased significantly in the hydrogel microgrooves of different widths (P<0.001). However, as the width of the hydrogel microgrooves increased, the number of cells with orientation angles <30° gradually decreased (79.9%, 62.3%, 54.7%, respectively), while the number of cells with orientation angles between 60°-90° increased (P<0.001). The cell bodies in the microgrooves were smaller and more rounded in shape. The cells were aligned along the direction of the microgrooves and corresponding changes occurred in the arrangement of the cell cytoskeleton. In the control group, cytoskeletal filaments were aligned in random directions, presenting an orientation angle of (45.5±3.7)°. Cytoskeletal filaments were distributed evenly within various orientation angles. However, in the 60 µm, 100 µm, and 140 µm hydrogel microgrooves, the orientation angles of the cytoskeletal filaments were significantly decreased, measuring (14.4±3.1)°, (24.7±3.5)°, and (31.9±3.3)°, respectively. The number of cytoskeletal filaments with orientation angles <30° significantly increased in hydrogel microgrooves of different widths (P<0.001). However, as the width of the hydrogel microgrooves increased, the number of cytoskeletal filaments with orientation angles <30° gradually decreased, while the number of cytoskeletal filaments with orientation angles between 60°-90° gradually increased (P<0.001). Conclusion: Hydrogel microgrooves can regulate the morphology and orientation of HUVECs and mimic to a certain extent the in vivo microenvironment of vascular endothelial cells, providing an experimental model that bears better resemblance to human physiology for the study of the unique physiological functions of vascular endothelial cells. Nonetheless, the molecular mechanism of spatial constraints on the morphology and the assembly of vascular endothelial cell needs to be further investigated.

A DNA-Micropatterned Surface for Propagating Biomolecular Signals by Positional on-off Assembly of Catalytic Nanocompartments.

Signal transduction is pivotal for the transfer of information between and within living cells. The composition and spatial organization of specified compartments are key to propagating soluble signals. Here, a high-throughput platform mimicking multistep signal transduction which is based on a geometrically defined array of immobilized catalytic nanocompartments (CNCs) that consist of distinct polymeric nanoassemblies encapsulating enzymes and DNA or enzymes alone is presented. The dual role of single entities or tandem CNCs in providing confined but communicating spaces for complex metabolic reactions and in protecting encapsulated compounds from denaturation is explored. To support a controlled spatial organization of CNCs, CNCs are patterned by means of DNA hybridization to a microprinted glass surface. Specifically, CNC-functionalized DNA microarrays are produced where individual reaction compartments are kept in close proximity by a distinct geometrical arrangement to promote effective communication. Besides a remarkable versatility and robustness, the most prominent feature of this platform is the reversibility of DNA-mediated CNC-anchoring which renders it reusable. Micropatterns of polymer-based nanocompartment assemblies offer an ideal scaffold for the development of the next generation responsive and communicative soft-matter analytical devices for applications in catalysis and medicine.

Controlling Morphology and Functions of Cardiac Organoids by Two-Dimensional Geometrical Templates.

Traditionally, tissue-specific organoids are generated as 3D aggregates of stem cells embedded in Matrigel or hydrogels, and the aggregates eventually end up a spherical shape and suspended in the matrix. Lack of geometrical control of organoid formation makes these spherical organoids limited for modeling the tissues with complex shapes. To address this challenge, we developed a new method to generate 3D spatial-organized cardiac organoids from 2D micropatterned human induced pluripotent stem cell (hiPSC) colonies, instead of directly from 3D stem cell aggregates. This new approach opens the possibility to create cardiac organoids that are templated by 2D non-spherical geometries, which potentially provides us a deeper understanding of biophysical controls on developmental organogenesis. Here, we designed 2D geometrical templates with quadrilateral shapes and pentagram shapes that had same total area but different geometrical shapes. Using this templated substrate, we grew cardiac organoids from hiPSCs and collected a series of parameters to characterize morphological and functional properties of the cardiac organoids. In quadrilateral templates, we found that increasing the aspect ratio impaired cardiac tissue 3D self-assembly, but the elongated geometry improved the cardiac contractile functions. However, in pentagram templates, cardiac organoid structure and function were optimized with a specific geometry of an ideal star shape. This study will shed a light on "organogenesis-by-design" by increasing the intricacy of starting templates from external geometrical cues to improve the organoid morphogenesis and functionality.

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Spatial Gradients of E-Cadherin and Fibronectin in TGF-ß1-Treated Epithelial Colonies Are Independent of Fibronectin Fibril Assembly.

Epithelial to Mesenchymal Transition (EMT) is a dynamic, morphogenetic process characterized by a phenotypic shift in epithelial cells towards a motile and often invasive mesenchymal phenotype. We have previously demonstrated that EMT is associated with an increase in assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils. We have also demonstrated that Transforming Growth Factor-ß1 (TGF-ß1) localizes to FN fibrils, and disruption of FN assembly or disruption of TGF-ß1 localization to FN fibrils attenuates EMT. Previous studies have shown that TGF-ß1 induces spatial gradients of EMT in mammary epithelial cells cultured on FN islands, with cells at free edges of the island preferentially undergoing EMT. In the current work, we sought to investigate: (a) whether FN fibril assembly is also spatially patterned in response to TGF-ß1, and (b) what effects FN fibril inhibition has on spatial gradients of E-Cadherin and FN fibrillogenesis. We demonstrate that mammary epithelial cells cultured on square micropatterns have fewer E-Cadherin-containing adherens junctions and assemble more FN fibrils at the periphery of the micropattern in response to increasing TGF-ß1 concentration, indicating that TGF-ß1 induces a spatial gradient of both E-Cadherin and FN fibrils. Inhibition of FN fibril assembly globally diminished E-Cadherin-containing adherens junctions and FN fibrillogenesis, but did not eliminate the spatial gradient of either. This suggests that global inhibition of FN reduces the degree of both FN fibrillogenesis and E-Cadherin-containing adherens junctions, but does not eliminate the spatial gradient of either, suggesting that spatial gradients of EMT and FN fibrillogenesis are influenced by additional factors.

Application and Development of Shape Memory Micro/Nano Patterns.

Shape memory polymers (SMPs) are a class of smart materials that change shape when stimulated by environmental stimuli. Different from the shape memory effect at the macro level, the introduction of micro-patterning technology into SMPs strengthens the exploration of the shape memory effect at the micro/nano level. The emergence of shape memory micro/nano patterns provides a new direction for the future development of smart polymers, and their applications in the fields of biomedicine/textile/micro-optics/adhesives show huge potential. In this review, the authors introduce the types of shape memory micro/nano patterns, summarize the preparation methods, then explore the imminent and potential applications in various fields. In the end, their shortcomings and future development direction are also proposed.

The Galapagos Chip Platform for High-Throughput Screening of Cell Adhesive Chemical Micropatterns.

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and -physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell-material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called "Galapagos chip" is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell-ECM interaction.

Simulated confluence on micropatterned substrates correlates responses regulating cellular differentiation.

While cells are known to behave differently based on the size of micropatterned islands, and this behavior is thought to be related to cell size and cell-cell contacts, the exact threshold for this difference between small and large islands is unknown. Furthermore, while cell size and cell-cell contacts can be easily manipulated on small islands, they are harder to measure and continually monitor on larger islands. To investigate this size threshold, and to explore cell size, cell-cell contacts, and differentiation, we use a previously established simulation to plan experiments and explain results that we could not explain from experiments alone. We use five seeding densities covering three orders of magnitude over 25-500 µm diameter islands to examine markers of proliferation and differentiation in bone marrow-derived mesenchymal cells (cell line). We show that osteogenic markers are most accurately described as a function of confluence for larger islands, but a function of time for smaller islands. We further show, using results of the simulation, that cell size and cell-cell contacts are also related to confluence on larger islands, but only cell-cell contacts are related to confluence on small islands. This study uses simulations to explain experimental results that could not be explained from experiments alone. Together, the simulations and experiments in this study show different differentiation patterns on large and small islands, and this simulation may be useful in planning future studies related to this study.

In vitro modeling of liver fibrosis in 3D microtissues using scalable micropatterning system.

Liver fibrosis is the late consequence of chronic liver inflammation which could eventually lead to cirrhosis, and liver failure. Among various etiological factors, activated hepatic stellate cells (aHSCs) are the major players in liver fibrosis. To date, various in vitro liver fibrosis models have been introduced to address biological and medical questions. Availability of traditional in vitro models could not fully recapitulate complicated pathology of liver fibrosis. The purpose of this study was to develop a simple and robust model to investigate the role of aHSCs on the progression of epithelial to mesenchymal transition (EMT) in hepatocytes during liver fibrogenesis. Therefore, we applied a micropatterning approach to generate 3D co-culture microtissues consisted of HepaRG and human umbilical cord endothelial cells (HUVEC) which co-cultured with inactivated LX-2 cells or activated LX-2 cells, respectively, as normal or fibrotic liver models in vitro. The result indicated that the activated LX-2 cells could induce EMT in HepaRG cells through activation of TGF-ß/SMAD signaling pathway. Besides, in the fibrotic microtissue, physiologic function of HepaRG cells attenuated compared to the control group, e.g., metabolic activity and albumin secretion. Moreover, our results showed that after treatment with Galunisertib, the fibrogenic properties decreased, in the term of gene and protein expression. In conclusion, it is proposed that aHSCs could lead to EMT in hepatocytes during liver fibrogenesis. Furthermore, the scalable micropatterning approach could provide enough required liver microtissues to prosper our understanding of the mechanisms involved in the progression of liver fibrosis as well as high throughput (HT) drug screening.

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity.

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts.

Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environments. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

Altering integrin engagement regulates membrane localization of Kir2.1 channels.

How ion channels localize and distribute on the cell membrane remains incompletely understood. We show that interventions that vary cell adhesion proteins and cell size also affect the membrane current density of inward-rectifier K+ channels (Kir2.1; encoded by KCNJ2) and profoundly alter the action potential shape of excitable cells. By using micropatterning to manipulate the localization and size of focal adhesions (FAs) in single HEK293 cells engineered to stably express Kir2.1 channels or in neonatal rat cardiomyocytes, we establish a robust linear correlation between FA coverage and the amplitude of Kir2.1 current at both the local and whole-cell levels. Confocal microscopy showed that Kir2.1 channels accumulate in membrane proximal to FAs. Selective pharmacological inhibition of key mediators of protein trafficking and the spatially dependent alterations in the dynamics of Kir2.1 fluorescent recovery after photobleaching revealed that the Kir2.1 channels are transported to the cell membrane uniformly, but are preferentially internalized by endocytosis at sites that are distal from FAs. Based on these results, we propose adhesion-regulated membrane localization of ion channels as a fundamental mechanism of controlling cellular electrophysiology via mechanochemical signals, independent of the direct ion channel mechanogating.

Reconstitution of cell migration at a glance.

Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of in vitro reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration in vivo These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.

Geometrical confinement controls the asymmetric patterning of brachyury in cultures of pluripotent cells.

Diffusible signals are known to orchestrate patterning during embryogenesis, yet diffusion is sensitive to noise. The fact that embryogenesis is remarkably robust suggests that additional layers of regulation reinforce patterning. Here, we demonstrate that geometrical confinement orchestrates the spatial organisation of initially randomly positioned subpopulations of spontaneously differentiating mouse embryonic stem cells. We use micropatterning in combination with pharmacological manipulations and quantitative imaging to dissociate the multiple effects of geometry. We show that the positioning of a pre-streak-like population marked by brachyury (T) is decoupled from the size of its population, and that breaking radial symmetry of patterns imposes polarised patterning. We provide evidence for a model in which the overall level of diffusible signals together with the history of the cell culture define the number of T+ cells, whereas geometrical constraints guide patterning in a multi-step process involving a differential response of the cells to multicellular spatial organisation. Our work provides a framework for investigating robustness of patterning and provides insights into how to guide symmetry-breaking events in aggregates of pluripotent cells.