Jamming of nephron-forming niches in the developing mouse kidney creates cyclical mechanical stresses.

Urinary collecting tubules form during kidney embryogenesis through the branching of the ureteric bud epithelium. A travelling mesenchyme niche of nephron progenitor cells caps each branching ureteric bud tip. These 'tip domain' niches pack more closely over developmental time and their number relates to nephron endowment at birth. Yet, how the crowded tissue environment impacts niche number and cell decision-making remains unclear. Here, through experiments and mathematical modelling, we show that niche packing conforms to physical limitations imposed by kidney curvature. We relate packing geometries to rigidity theory to predict a stiffening transition starting at embryonic day 15 in the mouse, validated by micromechanical analysis. Using a method to estimate tip domain 'ages' relative to their most recent branch events, we find that new niches overcome mechanical resistance as they branch and displace neighbours. This creates rhythmic mechanical stress in the niche. These findings expand our understanding of kidney development and inform engineering strategies for synthetic regenerative tissues.

Measurement of adhesion and traction of cells at high yield reveals an energetic ratchet operating during nephron condensation.

Developmental biology-inspired strategies for tissue-building have extraordinary promise for regenerative medicine, spurring interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here, we present multiplexed adhesion and traction of cells at high yield (MATCHY). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and a semiautomated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on a priori knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for rearranged during transfection (RET) tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We then use MATCHY to create a biophysical atlas of mouse embryonic kidney primary cells and identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an "energetic ratchet" that accounts for spatial trends in nephron progenitor cell condensation as they differentiate into early nephron structures, which we validate through agent-based computational simulation. MATCHY offers semiautomated cell biophysical characterization at >10,000-cell throughput, an advance benefiting fundamental studies and new synthetic tissue strategies for regenerative medicine.

Highly parallel production of designer organoids by mosaic patterning of progenitors.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures.

High-Throughput Assembly of Compositionally Controlled 3D Cell Communities for Developmental Engineering.

Cell patterning for 3D culture has increased our understanding of how cells interact among themselves and with their environment during tissue morphogenesis. Building cell communities from the bottom up with size and compositional control is invaluable for studies of morphological transitions. Here, we detail Photolithographic DNA-programmed Assembly of Cells (pDPAC). pDPAC uses a photoactive polyacrylamide gel substrate to capture single-stranded DNA on a 2D surface in large-scale, highly resolved patterns using the photomask technology. Cells are then functionalized with a complementary DNA strand, enabling cells to be temporarily adhered to distinct locations only where their complementary strand is patterned. These temporary 2D patterns can be transferred to extracellular matrix hydrogels for 3D culture of cells in biomimetic microenvironments. Use of a polyacrylamide substrate has advantages, including a simpler photolithography workflow, lower non-specific cell adhesion, and lower stiction to ECM hydrogels during release of patterned hydrogels. The protocol is equally applicable to large (cm)-scale patterns and repetitive arrays of smaller-scale cell interaction or migration experiments.

Measurement of adhesion and traction of cells at high yield (MATCHY) reveals an energetic ratchet driving nephron condensation.

Engineering of embryonic strategies for tissue-building has extraordinary promise for regenerative medicine. This has led to a resurgence in interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here we present MATCHY (multiplexed adhesion and traction of cells at high yield). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and an automated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on a priori knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for RET tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We go on to create a biophysical atlas of primary cells dissociated from the mouse embryonic kidney and use MATCHY to identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an 'energetic ratchet' that explains spatial nephron progenitor cell condensation from the niche as they differentiate, which we validate through agent-based computational simulation. MATCHY offers automated cell biophysical characterization at >104-cell throughput, a highly enabling advance for fundamental studies and new synthetic tissue design strategies for regenerative medicine.

Nephron progenitors rhythmically alternate between renewal and differentiation phases that synchronize with kidney branching morphogenesis.

The mammalian kidney achieves massive parallelization of function by exponentially duplicating nephron-forming niches during development. Each niche caps a tip of the ureteric bud epithelium (the future urinary collecting duct tree) as it undergoes branching morphogenesis, while nephron progenitors within niches balance self-renewal and differentiation to early nephron cells. Nephron formation rate approximately matches branching rate over a large fraction of mouse gestation, yet the nature of this apparent pace-maker is unknown. Here we correlate spatial transcriptomics data with branching 'life-cycle' to discover rhythmically alternating signatures of nephron progenitor differentiation and renewal across Wnt, Hippo-Yap, retinoic acid (RA), and other pathways. We then find in human stem-cell derived nephron progenitor organoids that Wnt/ß-catenin-induced differentiation is converted to a renewal signal when it temporally overlaps with YAP activation. Similar experiments using RA activation indicate a role in setting nephron progenitor exit from the naive state, the spatial extent of differentiation, and nephron segment bias. Together the data suggest that nephron progenitor interpretation of consistent Wnt/ß-catenin differentiation signaling in the niche may be modified by rhythmic activity in ancillary pathways to set the pace of nephron formation. This would synchronize nephron formation with ureteric bud branching, which creates new sites for nephron condensation. Our data bring temporal resolution to the renewal vs. differentiation balance in the nephrogenic niche and inform new strategies to achieve self-sustaining nephron formation in synthetic human kidney tissues.

Highly-parallel production of designer organoids by mosaic patterning of progenitors.

Human organoids are a promising approach for disease modeling and regenerative medicine. However, organoid variability and limited control over morphological outcomes remain significant challenges. Here we extend a DNA 'velcro' cell patterning approach, precisely controlling the number and ratio of human stem cell-derived progenitors contributing to nephron and mosaic nephron/ureteric bud organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints.

Rho/ROCK activity tunes cell compartment segregation and differentiation in nephron-forming niches.

Controlling the time and place of nephron formation in vitro would improve nephron density and connectivity in next-generation kidney replacement tissues. Recent developments in kidney organoid technology have paved the way to achieving self-sustaining nephrogenic niches in vitro. The physical and geometric structure of the niche are key control parameters in tissue engineering approaches. However, their relationship to nephron differentiation is unclear. Here we investigate the relationship between niche geometry, cell compartment mixing, and nephron differentiation by targeting the Rho/ROCK pathway, a master regulator of the actin cytoskeleton. We find that the ROCK inhibitor Y-27632 increases mixing between nephron progenitor and stromal compartments in native mouse embryonic kidney niches, and also increases nephrogenesis. Similar increases are also seen in reductionist mouse primary cell and human induced pluripotent stem cell (iPSC)-derived organoids perturbed by Y-27632, dependent on the presence of stromal cells. Our data indicate that niche organization is a determinant of nephron formation rate, bringing renewed focus to the spatial context of cell-cell interactions in kidney tissue engineering efforts.

Enhancing Single-Cell Western Blotting Sensitivity Using Diffusive Analyte Blotting and Antibody Conjugate Amplification.

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein content often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity Western blots on single cells (scWesterns) are attractive because they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by the affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address the sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in the limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates, which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 1000 molecules, a 120-fold improvement. This enables us to detect 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies compared to 84.5% of cells using in-gel detection. These results suggest the compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents─not previously accessible for in-gel use─for further signal amplification and detection of low-abundance targets.

Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification.

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 103 molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents - not previously accessible for in-gel use - for further signal amplification and detection of low abundance targets.

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology.

Tissue boundaries and interfaces are engines of morphogenesis in vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environment in vitro, fine-scale spatial control of cell positioning within tissue constructs remains an engineering challenge. To address this, we augment DNA "velcro" technology for selective patterning of ssDNA-labeled cells on mechanically defined photoactive polyacrylamide hydrogels. Hydrogels bearing photopatterned single-stranded DNA (ssDNA) features for cell capture are then co-functionalized with extracellular matrix (ECM) proteins to support subsequent adhesion of patterned tissues. ECM protein co-functionalization does not alter ssDNA pattern fidelity, cell capture, or hydrogel elastic stiffness. This approach enables mechanobiology studies and measurements of signaling activity at dynamic cell interfaces with precise initial patterning. Combining DNA velcro patterning and ECM functionalization provides independent control of initial cell placement, adhesion, and mechanics, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

The developing murine kidney actively negotiates geometric packing conflicts to avoid defects.

The physiological functions of several organs rely on branched epithelial tubule networks bearing specialized structures for secretion, gas exchange, or filtration. Little is known about conflicts in development between building enough tubules for adequate function and geometric constraints imposed by organ size. We show that the mouse embryonic kidney epithelium negotiates a physical packing conflict between increasing tubule tip numbers through branching and limited organ surface area. Through imaging of whole kidney explants, combined with computational and soft material modeling of tubule families, we identify six possible geometric packing phases, including two defective ones. Experiments in explants show that a radially oriented tension on tubule families is necessary and sufficient for them to switch to a vertical packing arrangement that increases surface tip density while avoiding defects. These results reveal developmental contingencies in response to physical limitations and create a framework for classifying congenital kidney defects.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Systematically quantifying morphological features reveals constraints on organoid phenotypes.

Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoid's morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoid's morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby canine kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

Bioengineering in vitro models of embryonic development.

Stem cell-based in vitro models of embryonic development have been established over the last decade. Such model systems recapitulate aspects of gametogenesis, early embryonic development, or organogenesis. They enable experimental approaches that have not been possible previously and have the potential to greatly reduce the number of animals required for research. However, each model system has its own limitations, with certain aspects, such as morphogenesis and spatiotemporal control of cell fate decisions, diverging from the in vivo counterpart. Targeted bioengineering approaches to provide defined instructive external signals or to modulate internal cellular signals could overcome some of these limitations. Here, we present the latest technical developments and discuss how bioengineering can further advance the optimization and external control of stem cell-based embryo-like structures (ELSs). In vitro models combined with sophisticated bioengineering tools will enable an even more in-depth analysis of embryonic development in the future.

Bioprinting for the Biologist.

Building tissues from scratch to explore entirely new cell configurations could revolutionize fundamental understanding in biology. Bioprinting is an emerging technology to do this. Although typically applied to engineer tissues for therapeutic tissue repair or drug screening, there are many opportunities for bioprinting within biology, such as for exploring cellular crosstalk or cellular morphogenesis. The overall goals of this Primer are to provide an overview of bioprinting with the biologist in mind, outline the steps in extrusion bioprinting (the most widely used and accessible technology), and discuss alternative bioprinting technologies and future opportunities for bioprinting in biology.

Guiding Cell Network Assembly using Shape-Morphing Hydrogels.

Forces and relative movement between cells and extracellular matrix (ECM) are crucial to the self-organization of tissues during development. However, the spatial range over which these dynamics can be controlled in engineering approaches is limited, impeding progress toward the construction of large, structurally mature tissues. Herein, shape-morphing materials called "kinomorphs" that rationally control the shape and size of multicellular networks are described. Kinomorphs are sheets of ECM that change their shape, size, and density depending on patterns of cell contractility within them. It is shown that these changes can manipulate structure-forming behaviors of epithelial cells in many spatial locations at once. Kinomorphs are built using a new photolithographic technology to pattern single cells into ECM sheets that are >10× larger than previously described. These patterns are designed to partially mimic the branch geometry of the embryonic kidney epithelial network. Origami-inspired simulations are then used to predict changes in kinomorph shapes. Last, kinomorph dynamics are shown to provide a centimeter-scale program that sets specific spatial locations in which ≈50 µm-diameter epithelial tubules form by cell coalescence and structural maturation. The kinomorphs may significantly advance organ-scale tissue construction by extending the spatial range of cell self-organization in emerging model systems such as organoids.

Quanti.us: a tool for rapid, flexible, crowd-based annotation of images.

We describe Quanti.us , a crowd-based image-annotation platform that provides an accurate alternative to computational algorithms for difficult image-analysis problems. We used Quanti.us for a variety of medium-throughput image-analysis tasks and achieved 10-50× savings in analysis time compared with that required for the same task by a single expert annotator. We show equivalent deep learning performance for Quanti.us-derived and expert-derived annotations, which should allow scalable integration with tailored machine learning algorithms.

Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme.

Many tissues fold into complex shapes during development. Controlling this process in vitro would represent an important advance for tissue engineering. We use embryonic tissue explants, finite element modeling, and 3D cell-patterning techniques to show that mechanical compaction of the extracellular matrix during mesenchymal condensation is sufficient to drive tissue folding along programmed trajectories. The process requires cell contractility, generates strains at tissue interfaces, and causes patterns of collagen alignment around and between condensates. Aligned collagen fibers support elevated tensions that promote the folding of interfaces along paths that can be predicted by modeling. We demonstrate the robustness and versatility of this strategy for sculpting tissue interfaces by directing the morphogenesis of a variety of folded tissue forms from patterns of mesenchymal condensates. These studies provide insight into the active mechanical properties of the embryonic mesenchyme and establish engineering strategies for more robustly directing tissue morphogenesis ex vivo.

Programmed synthesis of three-dimensional tissues.

Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis and disease in vitro. Here we describe DNA-programmed assembly of cells (DPAC), a method to reconstitute the multicellular organization of organoid-like tissues having programmed size, shape, composition and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide 'Velcro', allowing rapid, specific and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of organoid-like microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions and patterns of signaling heterogeneity on collective cell behaviors.