Confluent Epithelium Research Articles

A shape-driven reentrant jamming transition in confluent monolayers of synthetic cell-mimics

Many critical biological processes, like wound healing, require densely packed cell monolayers/tissues to transition from a jammed solid-like to a fluid-like state. Although numerical studies anticipate changes in the cell shape alone can lead to unjamming, experimental support for this prediction is not definitive because, in living systems, fluidization due to density changes cannot be ruled out. Additionally, a cell’s ability to modulate its motility only compounds difficulties since even in assemblies of rigid active particles, changing the nature of self-propulsion has non-trivial effects on the dynamics. Here, we design and assemble a monolayer of synthetic cell-mimics and examine their collective behaviour. By systematically increasing the persistence time of self-propulsion, we discovered a cell shape-driven, density-independent, re-entrant jamming transition. Notably, we observed cell shape and shape variability were mutually constrained in the confluent limit and followed the same universal scaling as that observed in confluent epithelia. Dynamical heterogeneities, however, did not conform to this scaling, with the fast cells showing suppressed shape variability, which our simulations revealed is due to a transient confinement effect of these cells by their slower neighbors. Our experiments unequivocally establish a morphodynamic link, demonstrating that geometric constraints alone can dictate epithelial jamming/unjamming.

Collagen Tubular Airway-on-Chip for Extended Epithelial Culture and Investigation of Ventilation Dynamics.

The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface culture at physiological shear stresses, the circular cross-section, and compliance of native airway walls have yet to be recapitulated. To overcome these limitations, a collagen tube-based airway model is presented. The lumen is lined with a confluent epithelium during two-week continuous perfusion with warm, humid air while presenting culture medium from the outside and compensating for evaporation. The model recapitulates human small airways in extracellular matrix composition and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing and mechanical ventilation, as well as their impactson the epithelium. A case study reveales increasing damage to the epithelium during repetitive collapse and reopening cycles as opposed to overdistension, suggesting expiratory flow resistance to reduce atelectasis. The model is expected to promote systematic comparisons between different clinically used ventilation strategies and, more broadly, to enhance human organ-on-a-chip platforms for a variety of tubular tissues.

Hydrodynamics and multiscale order in confluent epithelia.

We formulate a hydrodynamic theory of confluent epithelia: i.e. monolayers of epithelial cells adhering to each other without gaps. Taking advantage of recent progresses toward establishing a general hydrodynamic theory of p-atic liquid crystals, we demonstrate that collectively migrating epithelia feature both nematic (i.e. p = 2) and hexatic (i.e. p = 6) orders, with the former being dominant at large and the latter at small length scales. Such a remarkable multiscale liquid crystal order leaves a distinct signature in the system's structure factor, which exhibits two different power-law scaling regimes, reflecting both the hexagonal geometry of small cells clusters and the uniaxial structure of the global cellular flow. We support these analytical predictions with two different cell-resolved models of epithelia - i.e. the self-propelled Voronoi model and the multiphase field model - and highlight how momentum dissipation and noise influence the range of fluctuations at small length scales, thereby affecting the degree of cooperativity between cells. Our construction provides a theoretical framework to conceptualize the recent observation of multiscale order in layers of Madin-Darby canine kidney cells and pave the way for further theoretical developments.

3D vascularised proximal tubules-on-a-multiplexed chip model for enhanced cell phenotypes.

Modelling proximal tubule physiology and pharmacology is essential to understand tubular biology and guide drug discovery. To date, multiple models have been developed; however, their relevance to human disease has yet to be evaluated. Here, we report a 3D vascularized proximal tubule-on-a-multiplexed chip (3DvasPT-MC) device composed of co-localized cylindrical conduits lined with confluent epithelium and endothelium, embedded within a permeable matrix, and independently addressed by a closed-loop perfusion system. Each multiplexed chip contains six 3DvasPT models. We performed RNA-seq and compared the transcriptomic profile of proximal tubule epithelial cells (PTECs) and human glomerular endothelial cells (HGECs) seeded in our 3D vasPT-MCs and on 2D transwell controls with and without a gelatin-fibrin coating. Our results reveal that the transcriptional profile of PTECs is highly dependent on both the matrix and flow, while HGECs exhibit greater phenotypic plasticity and are affected by the matrix, PTECs, and flow. PTECs grown on non-coated Transwells display an enrichment of inflammatory markers, including TNF-a, IL-6, and CXCL6, resembling damaged tubules. However, this inflammatory response is not observed for 3D proximal tubules, which exhibit expression of kidney signature genes, including drug and solute transporters, akin to native tubular tissue. Likewise, the transcriptome of HGEC vessels resembled that of sc-RNAseq from glomerular endothelium when seeded on this matrix and subjected to flow. Our 3D vascularized tubule on chip model has utility for both renal physiology and pharmacology.

Couple stresses and discrete potentials in the vertex model of cellular monolayers.

The vertex model is widely used to simulate the mechanical properties of confluent epithelia and other multicellular tissues. This inherently discrete framework allows a Cauchy stress to be attributed to each cell, and its symmetric component has been widely reported, at least for planar monolayers. Here, we consider the stress attributed to the neighbourhood of each tricellular junction, evaluating in particular its leading-order antisymmetric component and the associated couple stresses, which characterise the degree to which individual cells experience (and resist) in-plane bending deformations. We develop discrete potential theory for localised monolayers having disordered internal structure and use this to derive the analogues of Airy and Mindlin stress functions. These scalar potentials typically have broad-banded spectra, highlighting the contributions of small-scale defects and boundary layers to global stress patterns. An affine approximation attributes couple stresses to pressure differences between cells sharing a trijunction, but simulations indicate an additional role for non-affine deformations.

A Platform for Co-Culture of Primary Human Colonic Epithelium With Anaerobic Probiotic Bacteria.

An in vitro platform was designed and optimized for the co-culture of probiotic anaerobic bacteria with a primary human colonic epithelium having a goal of assessing the anti-inflammatory impact of the probiotic bacteria. The device maintained a luminal O2 concentration at <1% while also supporting an oxygenated basal compartment at 10% for at least 72 h. Measurement of the transepithelial resistance of a confluent colonic epithelium showed high monolayer integrity while fluorescence assays demonstrated that the monolayer was comprised primarily of goblet cells and colonocytes, the two major differentiated cell subtypes of the colonic epithelium. High monolayer barrier function and viability were maintained during co-culture of the epithelium with the probiotic obligate anaerobe Anaerobutyricum hallii (A. hallii). Importantly the device supported a static co-culture of microbes and colonic epithelium mimicking the largely static or low flow conditions within the colonic lumen. A model inflamed colonic epithelium was generated by the addition of tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) to the basal and luminal epithelium sides, respectively. Co-culture of A. hallii with the LPS/TNF-α treated intestine diminished IL-8 secretion by ≥40% which could be mimicked by co-culture with the A. hallii metabolite butyrate. In contrast, co-culture of the inflamed epithelium with two strains of lactic acid-producing bacteria, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium adolescentis (B. adolescentis), did not diminish epithelial IL-8 secretion. Co-culture with colonic epithelial cells from different donors demonstrated a consistent anti-inflammatory effect by A. hallii, but distinct responses to co-culture with LGG and B. adolescentis. The demonstrated system offers a simple and easily adopted platform for examining the physiologic impact of alterations in the intestinal epithelium that occur in the presence of probiotic bacteria and their metabolites.

Cell-cell adhesion impacts epithelia response to substrate stiffness: Morphology and gene expression

Monolayer epithelial cells interact constantly with the substrate they reside on and their surrounding neighbors. As such, the properties of epithelial cells are profoundly governed by the mechanical and molecular cues that arise from both the substrate and contiguous cell neighbors. Although both cell-substrate and cell-cell interactions have been studied individually, these results are difficult to apply to native confluent epithelia, in which both jointly regulate the cell phenotype. Specifically, it remains poorly understood about the intertwined contributions from intercellular adhesion and substrate stiffness on cell morphology and gene expression, two essential microenvironment properties. Here, by adjusting the substrate modulus and altering the intercellular adhesion within confluent kidney epithelia, we found that cell-substrate and cell-cell interactions can mask each other's influence. For example, we found that epithelial cells exhibit an elongated morphological phenotype only when the substrate modulus and intercellular adhesions are both reduced, whereas their motility can be upregulated by either reduction. These results illustrate that combinatorial changes of the physical microenvironment are required to alter cell morphology and gene expression.

Multiphase field models for collective cell migration.

Confluent cell monolayers and epithelia tissues show remarkable patterns and correlations in structural arrangements and actively driven collective flows. We simulate these properties using multiphase field models. The models are based on cell deformations and cell-cell interactions and we investigate the influence of microscopic details to incorporate active forces on emerging phenomena. We compare four different approaches, one in which the activity is determined by a random orientation, one where the activity is related to the deformation of the cells, and two models with subcellular details to resolve the mechanochemical interactions underlying cell migration. The models are compared with respect to generic features, such as coordination number distribution, cell shape variability, emerging nematic properties, as well as vorticity correlations and flow patterns in large confluent monolayers and confinements. All results are compared with experimental data for a large variety of cell cultures. The appearing qualitative differences of the models show the importance of microscopic details and provide a route towards predictive simulations of patterns and correlations in cell colonies.

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Vertex Model instabilities in Epithelial Tissues Subject to Cellular Activity Or Applied Stress

Epithelial tissues, under many conditions, are subject to internal and external stresses. In particular, cells are continuously under active stresses due to processes like cell divisions, extrusions, neighboring rearrangements, cell growth, and contractions. in this work, we use a vertex model -- that has been recently used in computational descriptions of confluent epithelia, providing a direct connection with biophysical parameters -- to study the instabilities that appear when considering cell activity, such as modifications in their equilibrium perimeter, or external stresses, over a two-dimensional planar tissue. We show that for a wide range of parameters, the tissue becomes mechanically unstable as an effect of the cellular activity or the application of external stresses. Due to the confluent property, pure modes deformations are not allowed. instead, the tissue modes that become unstable are a coupling between pure-shear or deviatoric modes, with rotation and expansion modes. We perform analytic calculations considering a tissue made of regular hexagonal cells. Also, we realize numerical simulations using disordered tissues. We find that these kinds of tissues respond to the instabilities by increasing the variance of the flattening parameter of the cells, at short times, and by the appearance of non-convex cells, at longer times. We obtain an excellent agreement between the numerical simulations and the analytic results due to the homogenization of the tissue at long wavelengths. The presence of the predicted instabilities is a validation test to the vertex model to describe real epithelial tissues.

Solid-Liquid Transition of Deformable and Overlapping Active Particles.

Experiments and theory have shown that cell monolayers and epithelial tissues exhibit solid-liquid and glass-liquid transitions. These transitions are biologically relevant to our understanding of embryonic development, wound healing, and cancer. Current models of confluent epithelia have focused on the role of cell shape, with less attention paid to cell extrusion, which is key for maintaining homeostasis in biological tissue. Here, we use a multiphase field model to study the solid-liquid transition in a confluent monolayer of deformable cells. Cell overlap is allowed and provides a way for modeling the precursor for extrusion. When cells overlap rather than deform, we find that the melting transition changes from continuous to first order like, and that there is an intermittent regime close to the transition, where solid and liquid states alternate over time. By studying the dynamics of five- and sevenfold disclinations in the hexagonal lattice formed by the cell centers, we observe that these correlate with spatial fluctuations in the cellular overlap, and that cell extrusion tends to initiate near fivefold disclinations.

Force networks, torque balance and Airy stress in the planar vertex model of a confluent epithelium.

The vertex model is a popular framework for modelling tightly packed biological cells, such as confluent epithelia. Cells are described by convex polygons tiling the plane and their equilibrium is found by minimizing a global mechanical energy, with vertex locations treated as degrees of freedom. Drawing on analogies with granular materials, we describe the force network for a localized monolayer and derive the corresponding discrete Airy stress function, expressed for each N-sided cell as N scalars defined over kites covering the cell. We show how a torque balance (commonly overlooked in implementations of the vertex model) requires each internal vertex to lie at the orthocentre of the triangle formed by neighbouring edge centroids. Torque balance also places a geometric constraint on the stress in the neighbourhood of cellular trijunctions, and requires cell edges to be orthogonal to the links of a dual network that connect neighbouring cell centres and thereby triangulate the monolayer. We show how the Airy stress function depends on cell shape when a standard energy functional is adopted, and discuss implications for computational implementations of the model.

Renal reabsorption in 3D vascularized proximal tubule models

Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular-vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium-endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

Hypothesis: Caco-2 cell rotational 3D mechanogenomic turing patterns have clinical implications to colon crypts.

Colon crypts are recognized as a mechanical and biochemical Turing patterning model. Colon epithelial Caco‐2 cell monolayer demonstrated 2D Turing patterns via force analysis of apical tight junction live cell imaging which illuminated actomyosin meshwork linking the actomyosin network of individual cells. Actomyosin forces act in a mechanobiological manner that alters cell/nucleus/tissue morphology. We observed the rotational motion of the nucleus in Caco‐2 cells that appears to be driven by actomyosin during the formation of a differentiated confluent epithelium. Single‐ to multi‐cell ring/torus‐shaped genomes were observed prior to complex fractal Turing patterns extending from a rotating torus centre in a spiral pattern consistent with a gene morphogen motif. These features may contribute to the well‐described differentiation from stem cells at the crypt base to the luminal colon epithelium along the crypt axis. This observation may be useful to study the role of mechanogenomic processes and the underlying molecular mechanisms as determinants of cellular and tissue architecture in space and time, which is the focal point of the 4D nucleome initiative. Mathematical and bioengineer modelling of gene circuits and cell shapes may provide a powerful algorithm that will contribute to future precision medicine relevant to a number of common medical disorders.

Epithelial vertex models with active biochemical regulation of contractility can explain organized collective cell motility.

Collective motions of groups of cells are observed in many biological settings such as embryo development, tissue formation, and cancer metastasis. To effectively model collective cell movement, it is important to incorporate cell specific features such as cell size, cell shape, and cell mechanics, as well as active behavior of cells such as protrusion and force generation, contractile forces, and active biochemical signaling mechanisms that regulate cell behavior. In this paper, we develop a comprehensive model of collective cell migration in confluent epithelia based on the vertex modeling approach. We develop a method to compute cell-cell viscous friction based on the vertex model and incorporate RhoGTPase regulation of cortical myosin contraction. Global features of collective cell migration are examined by computing the spatial velocity correlation function. As active cell force parameters are varied, we found rich dynamical behavior. Furthermore, we find that cells exhibit nonlinear phenomena such as contractile waves and vortex formation. Together our work highlights the importance of active behavior of cells in generating collective cell movement. The vertex modeling approach is an efficient and versatile approach to rigorously examine cell motion in the epithelium.

Exosomes and Metabolic Function in Mice Exposed to Alternating Dark-Light Cycles Mimicking Night Shift Work Schedules.

Sleep is an important modulator of metabolic function. Disruptions of sleep in circadian rhythm are common in modern societies and are associated with increased risk of developing cardiometabolic disorders. Exosomes are ubiquitous extracellular vesicles that may play a mechanistic role in metabolic derangements. We hypothesized that alternating dark-light cycles mimicking shift work in mice would alter fecal microbiota and colonic epithelium permeability and alter plasma exosome cargo and metabolic function. C57BL/6 mice were randomly assigned to (i) control day light (CL), or (ii) inverted dark-light every 2 weeks for 8 weeks (IN). Body weight, fat mass and HOMA-IR were measured, along with Tregs, metabolic, and resident macrophages in visceral white adipose tissue (vWAT). Fecal water samples were incubated with confluent colonic epithelium cell cultures in electric cell-substrate impedance sensing (ECIS) arrays, and plasma exosomes were added to differentiated adipocytes and insulin-induced pAKT/AKT expression changes were assessed by western blots. Mice exposed to IN showed elevated HOMA-IR, and their fecal samples showed altered microbiota which promote increased permeability of the colonic epithelial cell barrier. Plasma exosomes decreased pAKT/AKT responses to exogenous insulin compared to CL, and altered expression of circadian clock genes. Inflammatory macrophages (Ly-6chigh) were increased in IN-exposed vWAT, while Tregs were decreased. Thus, gut microbiota and the cargo of plasma exosomes are altered by periodic shifts in environmental lighting, and effectively alter metabolic function, possibly via induction of systemic inflammation and altered clock expression in target tissues. Further exploration of exosomal miRNA signatures in shift workers and their putative metabolic organ cell targets appears warranted.

ARHGAP29 Mutation Is Associated with Abnormal Oral Epithelial Adhesions

Nonsyndromic cleft lip and/or palate (NSCL/P) is a prevalent birth defect of complex etiology. Previous studies identified mutations in ARHGAP29 associated with an increased risk for NSCL/P. To investigate the effects of ARHGAP29 in vivo, we generated a novel murine allele by inserting a point mutation identified in a patient with NSCL/P. This single-nucleotide variation of ARHGAP29 translates to an early nonsense mutation (K326X), presumably resulting in loss-of-function (LoF). Embryos from Arhgap29K326X/+ intercrosses were harvested at various time points. No homozygous Arhgap29K326X animals were found in the 45 analyzed litters, assessed as early as embryonic day 8.5 (e8.5). Coronal sectioning of e13.5 and e14.5 heads revealed that 59% of Arhgap29K326X/+ mice (n = 37) exhibited improper epithelial contact between developing oral structures, while none were observed in wild types (n = 10). In addition, Arhgap29K326X/+ embryos exhibited a significantly higher percentage of maxillary epithelium in contact with mandibular epithelium. Immunofluorescent analyses of the periderm and oral adhesions revealed the presence of Arhgap29 in periderm cells. These cells were p63 negative, keratin 17 positive, and keratin 6 positive and present at sites of adhesion, although occasionally disorganized. Oral adhesions did not appear to impair palatogenesis, as all analyzed Arhgap29K326X/+ embryos showed confluent palatal mesenchyme and epithelium at e18.5 (n = 16), and no mice were found with a cleft at birth. Collectively, our data demonstrate that ARHGAP29 is required for embryonic survival and that heterozygosity for LoF variants of Arhgap29 increases the incidence and length of oral adhesions at a critical time point during orofacial development. In conclusion, we validate the LoF nature of the human K326X mutation in vivo and reveal a previously unknown effect of Arhgap29 in murine craniofacial development.

Epithelial Cell Packing Induces Distinct Modes of Cell Extrusions

The control of tissue growth, which is a key to maintain the protective barrier function of the epithelium, depends on the balance between cell division and cell extrusion rates [1, 2]. Cells within confluent epithelial layers undergo cell extrusion, which relies on cell-cell interactions [3] and actomyosin contractility [4, 5]. Although it has been reported that cell extrusion is also dependent on cell density [6, 7], the contribution of tissue mechanics, which is tightly regulated by cell density [8-12], to cell extrusion is still poorly understood. By measuring the multicellular dynamics and traction forces, we show that changes in epithelial packing density lead to the emergence of distinct modes of cell extrusion. In confluent epithelia with low cell density, cell extrusion is mainly driven by the lamellipodia-based crawling mechanism in the neighbor non-dying cells in connection with large-scale collective movements. As cell density increases, cell motion is shown to slow down, and the role of a supracellular actomyosin cable formation and its contraction in the neighboring cells becomes the preponderant mechanism to locally promote cell extrusion. We propose that these two distinct mechanisms complement each other to ensure proper cell extrusion depending on the cellular environment. Our study provides a quantitative and robust framework to explain how cell density can influence tissue mechanics and in turn regulate cell extrusion mechanisms.

Polycystins and intercellular mechanotransduction: A precise dosage of polycystin 2 is necessary for alpha-actinin reinforcement of junctions upon mechanical stimulation

Polycystins 1 and 2, which are mutated in Autosomal Polycystic Kidney Disease, are involved in mechanotransduction through various mechanisms. In renal cells, polycystins not only have an important mechanotransductive role in primary cilia but are also present in intercellular contacts but their role there remains unclear. Here, we address the hypothesis that polycystins are involved in mechanotransduction via intercellular junctions, which would be expected to have consequences on tissue organization. We focused on the role of polycystin 2, which could be involved in mechanical organization at junctions either by its channel activity or by the direct recruitment of cytoskeleton components such as the F-actin cross-linker α-actinin. After mechanical stimulation of intercellular junctions in MDCK renal epithelial cells, α-actinin is rapidly recruited but this is inhibited upon overexpression of PC2 or the D509V mutant that lacks channel activity, and is also decreased upon PC2 silencing. This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive α-actinin recruitment at junctions. At the multicellular level, a change in PC2 expression was associated with changes in velocity in confluent epithelia and during wound healing together with a loss of orientation. This study suggests that the mechanosensitive regulation of cytoskeleton by polycystins in intercellular contacts may be important in the context of ADPKD.

Eviscerated Corneas as Tissue Source for Ex Vivo Expansion of Limbal Epithelial Cells on Platelet-Rich Plasma Gels

ABSTRACTPurpose/Aim of the study: To assess if corneal epithelium can be cultured ex vivo from corneas eviscerated due to irretrievable trauma, according to a cell culture method that made use of autologous platelet-rich plasma (A-PRP) as culture substrate. To compare corneal epithelium cultured ex vivo from corneas eviscerated following trauma using A-PRP combined with Dulbecco’s modified Eagles medium (DMEM), versus DMEM alone.Materials and methods: This was a laboratory case-controlled study of human corneal cells cultured in a mixture of A-PRP and DMEM, versus DMEM alone from six eviscerated corneas. A 100 explants were created, of which 50 explants were plated on A-PRP-gel construct combined with DMEM and 50 controls were placed in serum-free DMEM alone. Donor patients received systemic antibiotics prior to evisceration.Results: Confluent epithelium in monolayers could be cultured when donor limbal biopsies were placed in a mixture of A-PRP culture medium and DMEM. No growth was observed when corneas were placed in serum-free DMEM medium only (p < 0.05). No bacterial infection was observed in cultures.Conclusions: This study demonstrated that A-PRP is a viable and effective alternative to bovine serum for the ex vivo expansion of limbal epithelial cells. It also shows that eviscerated corneas are a viable source of donor tissue for this purpose in South Africa where access to tissue banks is limited.