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Advancing human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) technology will lead to significant progress ranging from disease modeling, to drug discovery, to regenerative tissue engineering. Yet, alongside these potential opportunities comes a critical challenge: attaining mature hiPSC-CM tissues. At present, there are multiple techniques to promote maturity of hiPSC-CMs including physical platforms and cell culture protocols. However, when it comes to making quantitative comparisons of functional behavior, there are limited options for reliably and reproducibly computing functional metrics that are suitable for direct cross-system comparison. In addition, the current standard functional metrics obtained from time-lapse images of cardiac microbundle contraction reported in the field (i.e., post forces, average tissue stress) do not take full advantage of the available information present in these data (i.e., full-field tissue displacements and strains). Thus, we present "MicroBundleCompute," a computational framework for automatic quantification of morphology-based mechanical metrics from movies of cardiac microbundles. Briefly, this computational framework offers tools for automatic tissue segmentation, tracking, and analysis of brightfield and phase contrast movies of beating cardiac microbundles. It is straightforward to implement, runs without user intervention, requires minimal input parameter setting selection, and is computationally inexpensive. In this paper, we describe the methods underlying this computational framework, show the results of our extensive validation studies, and demonstrate the utility of exploring heterogeneous tissue deformations and strains as functional metrics. With this manuscript, we disseminate "MicroBundleCompute" as an open-source computational tool with the aim of making automated quantitative analysis of beating cardiac microbundles more accessible to the community.

A better fundamental understanding of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has the potential to advance applications ranging from drug discovery to cardiac repair. Automated quantitative analysis of beating hiPSC-CMs is an important and fast developing component of the hiPSC-CM research pipeline. Here we introduce "Sarc-Graph," a computational framework to segment, track, and analyze sarcomeres in fluorescently tagged hiPSC-CMs. Our framework includes functions to segment z-discs and sarcomeres, track z-discs and sarcomeres in beating cells, and perform automated spatiotemporal analysis and data visualization. In addition to reporting good performance for sarcomere segmentation and tracking with little to no parameter tuning and a short runtime, we introduce two novel analysis approaches. First, we construct spatial graphs where z-discs correspond to nodes and sarcomeres correspond to edges. This makes measuring the network distance between each sarcomere (i.e., the number of connecting sarcomeres separating each sarcomere pair) straightforward. Second, we treat tracked and segmented components as fiducial markers and use them to compute the approximate deformation gradient of the entire tracked population. This represents a new quantitative descriptor of hiPSC-CM function. We showcase and validate our approach with both synthetic and experimental movies of beating hiPSC-CMs. By publishing Sarc-Graph, we aim to make automated quantitative analysis of hiPSC-CM behavior more accessible to the broader research community.

Mesenchymal stem cell (MSC) differentiation is mediated by soluble and physical cues. In this study, we investigated differentiation-induced transformations in MSC cellular and nuclear biophysical properties and queried their role in mechanosensation. Our data show that nuclei in differentiated bovine and human MSCs stiffen and become resistant to deformation. This attenuated nuclear deformation was governed by restructuring of Lamin A/C and increased heterochromatin content. This change in nuclear stiffness sensitized MSCs to mechanical-loading-induced calcium signaling and differentiated marker expression. This sensitization was reversed when the ‘stiff’ differentiated nucleus was softened and was enhanced when the ‘soft’ undifferentiated nucleus was stiffened through pharmacologic treatment. Interestingly, dynamic loading of undifferentiated MSCs, in the absence of soluble differentiation factors, stiffened and condensed the nucleus, and increased mechanosensitivity more rapidly than soluble factors. These data suggest that the nucleus acts as a mechanostat to modulate cellular mechanosensation during differentiation.

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of single fibronectin fibers enhanced the non-persistent migration of both Fnf/f and Fn-/- MEFs, the migration speed was increased for Fn-/- MEFs on plasma fibronectin fibers compared to Fnf/f MEFs. In contrast, the average speed was the same for all cells on collagen-coated Fn fibers. A Fn-FRET sensor revealed that fibronectin on average was more extended on the microtissue surface compared to fibronectin in the core. Gradients of collagen-to-fibronectin ratios and of the fraction of collagen-adsorbed to stretched fibrillar fibronectin conformations might thereby provide critical cell migration cues. This study highlights a dominant role for fibronectin in tissue morphogenesis and the development of tissue heterogeneities.

A well-established cascade of transcription factor (TF) activity orchestrates adipogenesis in response to chemical cues, yet how cell-intrinsic determinants of differentiation such as cell shape and/or seeding density inform this transcriptional program remain enigmatic. Here, we uncover a novel mechanism licensing transcription in human mesenchymal stem cells (hMSCs) adipogenically primed by confluence. Prior to adipogenesis, confluency promotes heterodimer recruitment of the bZip TFs C/EBPβ and ATF4 to a non-canonical C/EBP DNA sequence. ATF4 depletion decreases both cell-density-dependent transcription and adipocyte differentiation. Global profiling in hMSCs and a novel cell-free assay reveals that ATF4 requires C/EBPβ for genomic binding at a motif distinct from that bound by the C/EBPβ homodimer. Our observations demonstrate that C/EBPβ bridges the transcriptional programs in naïve, confluent cells and early differentiating pre-adipocytes. Moreover, they suggest that homo- and heterodimer formation poise C/EBPβ to execute diverse and stage-specific transcriptional programs by exploiting an expanded motif repertoire.

Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRASG12D mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRASG12D isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular in vitro platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma.

Cerebral cavernous malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system upon loss of the CCM multimeric protein complex. The endothelial cells within CCM lesions are characterized by overactive MEKK3 kinase and KLF2/4 transcription factor signaling, leading to pathological changes such as increased endothelial cell spreading and reduced junctional integrity. Concomitant to aberrant endothelial cell signaling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the central nervous system. Here, we adapted a three-dimensional microfluidic system to examine CCM1 deficient human micro-vessels in distinctive extracellular matrices. We validate that pathological hallmarks are maintained in this model. We further show that key genes responsible for homeostasis of hyaluronic acid, a major extracellular matrix component of the central nervous system, are dysregulated in CCM. Supplementing the matrix in our model with distinct forms of hyaluronic acid inhibits pathological cell spreading and rescues barrier function. Hyaluronic acid acts by dampening cell–matrix adhesion signaling in CCM, either downstream or in parallel of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signaling impact vascular malformations.

Epithelial-to-mesenchymal transition (EMT) is a complex process regulated at multiple molecular levels. Here, the authors implement an analytic framework - PAMAF - to integrate data from twelve distinct omics modalities, which they use to understand the molecular changes and regulation during EMT in vitro.

Introduction: Three dimensional engineered cardiac tissues (3D ECTs) have become indispensable as in vitro models to assess drug cardiotoxicity, a leading cause of failure in pharmaceutical development. A current bottleneck is the relatively low throughput of assays that measure spontaneous contractile forces exerted by millimeter-scale ECTs typically recorded through precise optical measurement of deflection of the polymer scaffolds that support them. The required resolution and speed limit the field of view to at most a few ECTs at a time using conventional imaging.Methods: To balance the inherent tradeoff among imaging resolution, field of view and speed, an innovative mosaic imaging system was designed, built, and validated to sense contractile force of 3D ECTs seeded on a 96-well plate. Results: The system performance was validated through real-time, parallel contractile force monitoring for up to 3 weeks. Pilot drug testing was conducted using isoproterenol.Discussion: The described tool increases contractile force sensing throughput to 96 samples per measurement; significantly reduces cost, time and labor needed for preclinical cardiotoxicity assay using 3D ECT. More broadly, our mosaicking approach is a general way to scale up image-based screening in multi-well formats.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Here authors show that force- FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair.

Abstract Interactions between cardiac myofibroblasts and myocytes may slow conduction and generate spontaneous beating in fibrosis, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the extent of myofibroblast-myocyte electrical conductance in a syncytium is unknown. In this neonatal rat study, cardiac myofibroblasts were transduced with Channelrhodopsin-2, which allowed acute and selective increase of myofibroblast current, and plated on top of cardiomyocytes. Optical mapping revealed significantly decreased conduction velocity (− 27 ± 6%, p

Abstract The structural and functional maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential for pharmaceutical testing, disease modeling, and ultimately therapeutic use. Multicellular 3D-tissue platforms have improved the functional maturation of hiPSC-CMs, but probing cardiac contractile properties in a 3D environment remains challenging, especially at depth and in live tissues. Using small-angle X-ray scattering (SAXS) imaging, we show that hiPSC-CMs matured and examined in a 3D environment exhibit a periodic spatial arrangement of the myofilament lattice, which has not been previously detected in hiPSC-CMs. The contractile force is found to correlate with both the scattering intensity (R 2 = 0.44) and lattice spacing (R 2 = 0.46). The scattering intensity also correlates with lattice spacing (R 2 = 0.81), suggestive of lower noise in our structural measurement than in the functional measurement. Notably, we observed decreased myofilament ordering in tissues with a myofilament mutation known to lead to hypertrophic cardiomyopathy (HCM). Our results highlight the progress of human cardiac tissue engineering and enable unprecedented study of structural maturation in hiPSC-CMs.

Summary: Spatial regulation of angiogenesis is important for the generation of functional engineered vasculature in regenerative medicine. The Notch ligands Jag1 and Dll4 show distinct expression patterns in endothelial cells and, respectively, promote and inhibit endothelial sprouting. Therefore, patterns of Notch ligands may be utilized to spatially control sprouting, but their potential and the underlying mechanisms of action are unclear. Here, we coupled in vitro and in silico models to analyze the ability of micropatterned Jag1 and Dll4 ligands to spatially control endothelial sprouting. Dll4 patterns, but not Jag1 patterns, elicited spatial control. Computational simulations of the underlying signaling dynamics suggest that different timing of Notch activation by Jag1 and Dll4 underlie their distinct ability to spatially control sprouting. Hence, Dll4 patterns efficiently direct the sprouts, whereas longer exposure to Jag1 patterns is required to achieve spatial control. These insights in sprouting regulation offer therapeutic handles for spatial regulation of angiogenesis.

In vitro models of the human mammary gland have struggled to mimic the 3D morphogenic processes that occur in vivo. Here the authors develop a 3D microfluidic platform of a vascularized human mammary duct that simulates diverse morphogenic transitions and paracrine crosstalk.

Abstract Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch‐dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue.

The formation of healthy vascularized granulation tissue is essential for rapid wound closure and the prevention of chronic wounds in humans, yet how endothelial cells and fibroblasts coordinate during this process has been difficult to study. Here, we have developed an in vitro system that reveals how human endothelial and stromal cells in a 3D matrix respond during wound healing and granulation tissue formation. By creating incisions in engineered cultures composed of human umbilical vein endothelial cells and human lung fibroblasts embedded within a 3D matrix, we observed that these tissues are able to close the wound within approximately 4 days. Live tracking of cells during wound closure revealed that the process is mediated primarily by fibroblasts. The fibroblasts migrate circumferentially around the wound edge during early phases of healing, while contracting the wound. The fibroblast-derived matrix is, then, deposited into the void, facilitating fibroblast migration toward the wound center and filling of the void. Interestingly, the endothelial cells remain at the periphery of the wound rather than actively sprouting into the healing region to restore the vascular network. This study captures the dynamics of endothelial and fibroblast-mediated closure of three-dimensional wounds, which results in the repopulation of the wound with the cell-derived extracellular matrix representative of early granulation tissue, thus presenting a model for future studies to investigate factors regulating vascularized granulation tissue formation.

Summary: Precise measurement of action potentials (APs) is needed to observe electrical activity and cellular communication within cardiac tissue. Voltage-sensitive dyes (VSDs) are traditionally used to measure cardiac APs; however, they require acute chemical addition that prevents chronic imaging. Genetically encoded voltage indicators (GEVIs) enable long-term studies of APs without the need of chemical additions, but current GEVIs used in cardiac tissue exhibit poor kinetics and/or low signal to noise (SNR). Here, we demonstrate the use of Archon1, a recently developed GEVI, in hiPSC-derived cardiomyocytes (CMs). When expressed in CMs, Archon1 demonstrated fast kinetics comparable with patch-clamp electrophysiology and high SNR significantly greater than the VSD Di-8-ANEPPS. Additionally, Archon1 enabled monitoring of APs across multiple cells simultaneously in 3D cardiac tissues. These results highlight Archon1's capability to investigate the electrical activity of CMs in a variety of applications and its potential to probe functionally complex in vitro models, as well as in vivo systems. : Biotechnology; Bioelectronics; Technical Aspects of Cell Biology; Electronic Materials Subject Areas: Biotechnology, Bioelectronics, Technical Aspects of Cell Biology, Electronic Materials

Summary: Here, we show that microfluidic organ-on-a-chip (organ chip) cell culture technology can be used to create in vitro human orthotopic models of non-small-cell lung cancer (NSCLC) that recapitulate organ microenvironment-specific cancer growth, tumor dormancy, and responses to tyrosine kinase inhibitor (TKI) therapy observed in human patients in vivo. Use of the mechanical actuation functionalities of this technology revealed a previously unknown sensitivity of lung cancer cell growth, invasion, and TKI therapeutic responses to physical cues associated with breathing motions, which appear to be mediated by changes in signaling through epidermal growth factor receptor (EGFR) and MET protein kinase. These findings might help to explain the high level of resistance to therapy in cancer patients with minimal residual disease in regions of the lung that remain functionally aerated and mobile, in addition to providing an experimental model to study cancer persister cells and mechanisms of tumor dormancy in vitro. : Hassell et al. demonstrate that, when human lung cancer cells are grown within organ-on-a-chip culture devices that mimic lung structure and function, tumor cells recapitulate tumor growth and invasion patterns, as well as responses to therapy, observed in human patients. They also discover that breathing motions influence these responses. Keywords: lung cancer, microfluidic, organ-on-chip, invasion, persister cell, tyrosine kinase inhibitor, EGFR inhibitor, mechanical, chemotherapy, mechanobiology

The fabrication of vascularized 3D tissues requires an understanding of how material properties govern endothelial cell invasion into the surrounding matrix. Here the authors integrate a non-swelling synthetic hydrogel with a microfluidic device to study chemokine gradient-driven angiogenic sprouting and find that matrix degradability modulates the collectivity of cell migration.

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling.

Planar in vitromodels for wound closure stress the role of lamellipodial protrusions and purse-string contraction. Here the authors develop a 3D biomimetic model for tissue repair and show a mode of stromal closure that relies on whole tissue deformations, cell migration and matrix deposition.

Physical features of the extracellular matrix (ECM) heavily influence cell migration strategies and efficiency. Migration in and on fibrous ECMs is of significant physiologic importance, but limitations in the ability to experimentally define the diameter, density, and alignment of native ECMs in vitro have hampered our understanding of how these properties affect this basic cell function. Here, we designed a high-throughput in vitro platform that models fibrous ECM as collections of lines of cell-adhesive fibronectin on a flat surface to eliminate effects of dimensionality and topography. Using a microcontact printing approach to orthogonally vary line alignment, density, and size, we determined each factor's individual influence on NIH3T3 fibroblast migration. High content imaging and statistical analyses revealed that ECM alignment is the most critical parameter in influencing cell morphology, polarization, and migratory behavior. Specifically, increasing ECM alignment led cells to adopt an elongated uniaxial morphology and migrate with enhanced speed and persistence. Intriguingly, migration speeds were tightly correlated with the organization of focal adhesions, where cells with the most aligned adhesions migrated fastest. Highly organized focal adhesions and associated actin stress fibers appeared to define the number and location of protrusive fronts, suggesting that ECM alignment influences active Rac1 localization. Utilizing a novel microcontact-printing approach that lacks confounding influences of substrate dimensionality, mechanics, or differences in the adhesive area, this work highlights the effect of ECM alignment on orchestrating the cytoskeletal machinery that governs directed uniaxial cell migration.

In contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs) in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK)-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness- and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.

Summary It has previously been demonstrated that cell shape can influence commitment of human bone marrow-derived mesenchymal stem cells (hBMCs) to adipogenic, osteogenic, chondrogenic, and other lineages. Human periosteum-derived cells (hPDCs) exhibit multipotency similar to hBMCs, but hPDCs may offer enhanced potential for osteogenesis and chondrogenesis given their apparent endogenous role in bone and cartilage repair in vivo. Here, we examined whether hPDC differentiation is regulated by adhesive and mechanical cues comparable to that reported for hBMC differentiation. When cultured in the appropriate induction media, hPDCs at high cell seeding density demonstrated enhanced levels of adipogenic or chondrogenic markers as compared with hPDCs at low cell seeding density. Cell seeding density correlated inversely with projected area of cell spreading, and directly limiting cell spreading with micropatterned substrates promoted adipogenesis or chondrogenesis while substrates promoting cell spreading supported osteogenesis. Interestingly, cell seeding density influenced differentiation through both changes in cell shape and non-shape-mediated effects: density-dependent adipogenesis and chondrogenesis were regulated primarily by cell shape whereas non-shape effects strongly influenced osteogenic potential. Inhibition of cytoskeletal contractility by adding the Rho kinase inhibitor Y27632 further enhanced adipogenic differentiation and discouraged osteogenic differentiation of hPDCs. Together, our results suggest that multipotent lineage decisions of hPDCs are impacted by cell adhesive and mechanical cues, though to different extents than hBMCs. Thus, future studies of hPDCs and other primary stem cell populations with clinical potential should consider varying biophysical metrics for more thorough optimization of stem cell differentiation.

Loss of function mutations in the nuclear inner membrane protein, emerin, cause X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). X-EDMD is characterized by contractures of major tendons, skeletal muscle weakening and wasting, and cardiac conduction system defects. The transcription factor Lmo7 regulates muscle- and heart-relevant genes and is inhibited by binding to emerin, suggesting Lmo7 misregulation contributes to EDMD disease. Lmo7 associates with cell adhesions and shuttles between the plasma membrane and nucleus, but the regulation and biological consequences of this dual localization were unknown. We report endogenous Lmo7 also associates with focal adhesions in cells, and both co-localizes and co-immunoprecipitates with p130Cas, a key signaling component of focal adhesions. Lmo7 nuclear localization and transcriptional activity increased significantly in p130Cas-null MEFs, suggesting Lmo7 is negatively regulated by p130Cas-dependent association with focal adhesions. These results support EDMD models in which Lmo7 is a downstream mediator of integrin-dependent signaling that allows tendon cells and muscles to adapt to and withstand mechanical stress.