WNT-SHH Antagonism Specifies and Expands Stem Cells prior to Niche Formation.

Adult stem cell (SC) maintenance and differentiation are known to depend on signals received from the niche. Here, however, we demonstrate a mechanism for SC specification and regulation that is niche independent. Using immunofluorescence, live imaging, genetics, cell-cycle analyses, in utero lentiviral transduction, and lineage-tracing, we show that in developing hair buds, SCs are born from asymmetric divisions that differentially display WNT and SHH signaling. Displaced WNT(lo) suprabasal daughters become SCs that respond to paracrine SHH and symmetrically expand. By contrast, basal daughters remain WNT(hi). They express but do not respond to SHH and hence maintain slow-cycling, asymmetric divisions. Over time, they become short-lived progenitors, generating differentiating daughters rather than SCs. Thus, in contrast to an established niche that harbors a fixed SC pool whose expelled progeny differentiate, asymmetric divisions first specify and displace early SCs into an environment conducive to expansion and later restrict their numbers by switching asymmetric fates.

Publisher Correction: Mechanics of a multilayer epithelium instruct tumour architecture and function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanics of a multilayer epithelium instruct tumour architecture and function.

Loss of normal tissue architecture is a hallmark of oncogenic transformation1. In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis2. However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.

Physical and chemical microenvironmental cues orthogonally control the degree and duration of fibrosis-associated epithelial-to-mesenchymal transitions.

Increased tissue stiffness and epithelial-to-mesenchymal transitions (EMTs) are two seemingly discrete hallmarks of fibrotic diseases. Despite recent findings highlighting the influence of tissue mechanical properties on cell phenotype, it remains unclear what role increased tissue stiffness has in the regulation of previously reported fibronectin-mediated EMTs associated with pulmonary fibrosis. Nano-indentation testing of lung interstitial spaces showed that in vivo cell-level Young's moduli increase with the onset of fibrosis from ∼2 to ∼17 kPa. In vitro, we found that stiff, but not soft, fibronectin substrates induce EMT, a response dependent on cell contraction-mediated integrin activation of TGFß. Activation or suppression of cell contractility with exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicate that the effect of cell contractility is dose- and time-dependent. In response to low levels of TGFß on soft surfaces, either added exogenously or produced through thrombin-induced contraction, cells will initiate the EMT programme, but upon removal revert to an epithelial phenotype. These results identify matrix stiffness and/or cell contractility as critical targets for novel therapeutics for fibrotic diseases.

Synergistic effects of particulate matter and substrate stiffness on epithelial-to-mesenchymal transition.

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD*), and tumorigenesis, have been increasing steadily over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that the lung "precursor cell"--the alveolar type II (ATII) epithelial cell--is central in the initiation and progression of pulmonary fibrosis. Specifically, ATII cells have been shown (Iwano et al. 2002) to be capable of undergoing an epithelial-to-mesenchymal transition (EMT). EMT, the de-differentiation of an epithelial cell into a mesenchymal cell, has been theorized to increase the number of extracellular matrix (ECM)-secreting mesenchymal cells, perpetuating fibrotic conditions and resulting in increased lung tissue stiffness. In addition, increased exposure to pollution and inhalation of particulate matter (PM) have been shown to be highly correlated with an increased incidence of pulmonary fibrosis. Although both of these events are involved in the progression of pulmonary fibrosis, the relationship between tissue stiffness, exposure to PM, and the initiation and course of EMT remains unclear. The hypothesis of this study was twofold: 1. That alveolar epithelial cells cultured on increasingly stiff substrates become increasingly contractile, leading to enhanced transforming growth factor beta (TGF-ß) activation and EMT; and 2. That exposure of alveolar epithelial cells to PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5; also known as fine PM) results in enhanced cell contractility and EMT. Our study focused on the relationship between the micromechanical environment and external environmental stimuli on the phenotype of alveolar epithelial cells. This relationship was explored by first determining how increased tissue stiffness affects the regulation of fibronectin (Fn)-mediated EMT in ATII cells in vitro. We cultured ATII cells on substrates of increasing stiffness and evaluated changes in cell contractility and EMT. We found that stiff, but not soft, Fn substrates were able to induce EMT and that this event depended on a contractile phenotype of the cell and the subsequent activation of TGF-ß. In addition, we were able to show that activation or suppression of cell contractility by way of exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicated that the effect on cell contractility is dose- and time-dependent. In response to low levels of TGF-ß on soft surfaces, either added exogenously or produced through contraction induced by the stiffness agonist thrombin, cells initiate EMT; on removal of the TGF-ß, they revert to an epithelial phenotype. Overall, the results from this first part of our study identified matrix stiffness or cell contractility as critical targets for the control of EMT in fibrotic diseases. For the second part of our study, we wanted to investigate whether exposure to PM2.5, which might have higher toxicity than coarser PM because of its small size and large surface-to-mass ratio, altered the observed stiffness-mediated EMT. Again, we cultured ATII cells on increasingly stiff substrates with or without the addition of three concentrations of PM2.5. We found that exposure to PM2.5 was involved in increased stiffness-mediated EMT, as shown by increases in mesenchymal markers, cell contractility, and TGF-ß activation. Most notably, on substrates with an elastic modulus (E) of 8 kilopascals (kPa), a physiologically relevant range for pulmonary fibrosis, the addition of PM2.5 resulted in increased mesenchymal cells and EMT; these were not seen in the absence of the PM2.5. Overall, this study showed that there is a delicate balance between substrate stiffness, TGF-ß, and EMT. Furthermore, we showed that exposure to PM2.5 is able to further mediate this interaction. The higher levels of EMT seen with exposure to PM2.5 might have been a result of a positive feedback loop, in which enhanced exposure to PM2.5 through the loss of cell-cell junctions during the initial stages of EMT led to the cells being more susceptible to the effects of surrounding immune cells and inflammatory signals that can further activate TGF-ß and drive additional EMT progression. Overall, our work--showing increased cell contractility, TGF-ß activation, and EMT in response to substrate stiffness and PM2.5 exposure--highlights the importance of both the micromechanical and biochemical environments in lung disease. These findings suggest that already-fibrotic tissue might be more susceptible to further damage than healthy tissue when exposed to PM2.5.

Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.

RATIONALE: Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis. OBJECTIVES: To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs. METHODS: Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured. MEASUREMENTS AND MAIN RESULTS: We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tissue. We demonstrate that lung fibroblasts reseeded into acellular lung matrices can be subsequently assayed using conventional protocols; in this manner we show that fibrotic matrices clearly promote transforming growth factor-ß-independent myofibroblast differentiation compared with normal matrices. Furthermore, comprehensive analysis of acellular matrix ECM details significant compositional differences between normal and fibrotic lungs, paving the way for further study of novel hypotheses. CONCLUSIONS: This methodology is expected to allow investigation of important ECM-based hypotheses in human tissues and permits future scientific exploration in an organ- and disease-specific manner.

Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction.

The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.

The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.

Liquid-liquid phase separation drives skin barrier formation.

At the body surface, skin's stratified squamous epithelium is challenged by environmental extremes. The surface of the skin is composed of enucleated, flattened surface squames. They derive from underlying, transcriptionally active keratinocytes that display filaggrin-containing keratohyalin granules (KGs) whose function is unclear. Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. The dynamics of phase separation governed terminal differentiation and were disrupted by human skin barrier disease-associated mutations. We used fluorescent sensors to investigate endogenous phase behavior in mice. Phase transitions during epidermal stratification crowded cellular spaces with liquid-like KGs whose coalescence was restricted by keratin filament bundles. We imaged cells as they neared the skin surface and found that environmentally regulated KG phase dynamics drive squame formation. Thus, epidermal structure and function are driven by phase-separation dynamics.

αvß3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis.

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvß3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvß3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvß3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvß3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.

Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction.

Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive αvß3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1's glycophosphatidylinositol tether. Disruption of Thy-1-αvß3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis.

Dynamic catch of a Thy-1-α5ß1+syndecan-4 trimolecular complex.

Cancer cell adhesion to the vascular endothelium is a critical step of tumour metastasis. Endothelial surface molecule Thy-1 (CD90) is implicated in the metastatic process through its interactions with integrins and syndecans. However, how Thy-1 supports cell-cell adhesion in a dynamic mechanical environment is not known. Here we show that Thy-1 supports ß1 integrin- and syndecan-4 (Syn4)-mediated contractility-dependent mechanosignalling of melanoma cells. At the single-molecule level, Thy-1 is capable of independently binding α5ß1 integrin and syndecan-4 (Syn4) receptors. However, in the presence of both α5ß1 and Syn4, the two receptors bind cooperatively to Thy-1, to form a trimolecular complex. This trimolecular complex displays a unique phenomenon we coin 'dynamic catch', characterized by abrupt bond stiffening followed by the formation of catch bonds, where force prolongs the bond lifetime. Thus, we reveal a new class of trimolecular interactions where force strengthens the synergistic binding of two co-receptors and modulates downstream mechanosignalling.

One primer to rule them all: universal primer that adds BBa_B0034 ribosomal binding site to any coding standard 10 BioBrick.

Here, we present a universal, simple, efficient, and reliable way to add small BioBrick parts to any BioBrick via PCR that is compatible with BioBrick assembly standard 10. As a proof of principle, we have designed a universal primer, rbs_B0034, that contains a ribosomal binding site (RBS; BBa_B0034) and that can be used in PCR to amplify any coding BioBrick that starts with ATG. We performed test PCRs with rbs_B0034 on 31 different targets and found it to be 93.6% efficient. Moreover, when supplemented with a complementary primer, addition of RBS can be accomplished via whole plasmid site-directed mutagenesis, thus reducing the time required for further assembly of composite parts. The described method brings simplicity to the addition of small parts, such as regulatory elements to existing BioBricks. The final product of the PCR assembly is indistinguishable from the standard or 3A BioBrick assembly.

The modulation of cardiac progenitor cell function by hydrogel-dependent Notch1 activation.

Myocardial infarction is the leading cause of death worldwide and phase I clinical trials utilizing cardiac progenitor cells (CPCs) have shown promising outcomes. Notch1 signaling plays a critical role in cardiac development and in the survival, cardiogenic lineage commitment, and differentiation of cardiac stem/progenitor cells. In this study, we functionalized self-assembling peptide (SAP) hydrogels with a peptide mimic of the Notch1 ligand Jagged1 (RJ) to evaluate the therapeutic benefit of CPC delivery in the hydrogels in a rat model of myocardial infarction. The behavior of CPCs cultured in the 3D hydrogels in vitro including gene expression, proliferation, and growth factor production was evaluated. Interestingly, we observed Notch1 activation to be dependent on hydrogel polymer density/stiffness with synergistic increase in presence of RJ. Our results show that RJ mediated Notch1 activation depending on hydrogel concentration differentially regulated cardiogenic gene expression, proliferation, and growth factor production in CPCs in vitro. In rats subjected to experimental myocardial infarction, improvement in acute retention and cardiac function was observed following cell therapy in RJ hydrogels compared to unmodified or scrambled peptide containing hydrogels. This study demonstrates the potential therapeutic benefit of functionalizing SAP hydrogels with RJ for CPC based cardiac repair.

Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells.

The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses.

Polyketal microparticles for therapeutic delivery to the lung.

Inflammation in the setting of interstitial lung disease (ILD) occurs in the distal alveolar spaces of the lung, which presents significant challenges for therapeutic delivery. The development of aerosolizable microparticles from non-immunogenic polymers is needed to enable the clinical translation of numerous experimental therapeutics that require localization to the deep lung and repeated delivery for optimal efficacy. Polyketals (PK), a family of polymers, have several unique properties that make them ideal for lung delivery, specifically their hydrolysis into non-acidic, membrane-permeable compounds and their capacity to form microparticles with the aerodynamic properties needed for aerosolization. In this study, we tested the lung biocompatibility of microparticles created from a polyketal polymer, termed PK3, following intratracheal instillation in comparison to commonly used PLGA microparticles. We furthermore tested the initial efficacy of PK3 microparticles to encapsulate and effectively deliver active superoxide dismutase (SOD), a free radical scavenging enzyme, in a model of lung fibrosis. Our findings indicate that PK3 microparticles display no detectable level of alveolar or airway inflammation, whereas PLGA induced a small inflammatory response. Furthermore, SOD-loaded into PK3 microparticles maintained its activity upon release and, when delivered via PK3 microparticles, inhibited the extent of lung fibrosis.

Interfacial energetics of protein adsorption from aqueous buffer to surfaces with varying hydrophilicity.

Adsorption isotherms constructed from time-and-concentration-dependent advancing contact angles thetaa show that the profound biochemical diversity among ten different blood proteins with molecular weight spanning 10-1000 kDa has little discernible effect on the amount adsorbed from aqueous phosphate-buffered saline (PBS) solution after 1 h contact with a particular test surface selected from the full range of observable water wettability (as quantified by PBS adhesion tension tauoa=gammaolv cos thetaoa; where gammaolv is the liquid-vapor interfacial tension and thetaoa is the advancing PBS contact angle). The maximum advancing spreading pressure, Pimaxa, determined from adsorption isotherms decreases systematically with tauoa for methyl-terminated self-assembled monolayers (CH3 SAM, tauo=-15 mN/m), polystyrene spun-coated onto electronic-grade SiOx wafers (PS, tauo=7.2 mN/m), aminopropyltriethoxysilane-treated SiOx surfaces (APTES, tauo = 42 mN/m), and fully water wettable SiOx (tauo=72 mN/m). Likewise, the apparent Gibbs' surface excess [Gammasl-Gammasv], which measures the difference in the amount of protein adsorbed Gamma (mol/cm2) at solid-vapor (SV) and solid-liquid (SL) interfaces, decreases with tauo from maximal values measured on the CH3 SAM surface through zero (no protein adsorption in excess of bulk solution concentration) near tauo=30 mN/m (thetaa=65 degrees). These latter results corroborate the conclusion drawn from independent studies that water is too strongly bound to surfaces with tauo>or=30 mN/m to be displaced by adsorbing protein and that, as a consequence, protein does not accumulate within the interfacial region of such surfaces at concentrations exceeding that of bulk solution ([Gammasl-Gammasv]=0 at tauo=30 mN/m). Results are collectively interpreted to mean that water controls protein adsorption to surfaces and that the mechanism of protein adsorption can be understood from this perspective for a diverse set of proteins with very different amino acid compositions.