Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro
Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro

Abstract: Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

Originally Published in: Biomaterials Science (2023) (Link to Paper)

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Curi CytostretcherArianna KieserSkeletal Muscle Cells
Deep learning detects cardiotoxicity in a high-content screen with induced pluripotent stem cell-derived cardiomyocytes
Deep learning detects cardiotoxicity in a high-content screen with induced pluripotent stem cell-derived cardiomyocytes

Drug-induced cardiotoxicity and hepatotoxicity are major causes of drug attrition. To decrease late-stage drug attrition, pharmaceutical and biotechnology industries need to establish biologically relevant models that use phenotypic screening to detect drug-induced toxicity in vitro. In this study, we sought to rapidly detect patterns of cardiotoxicity using high-content image analysis with deep learning and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We screened a library of 1280 bioactive compounds and identified those with potential cardiotoxic liabilities in iPSC-CMs using a single-parameter score based on deep learning. Compounds demonstrating cardiotoxicity in iPSC-CMs included DNA intercalators, ion channel blockers, epidermal growth factor receptor, cyclin-dependent kinase, and multi-kinase inhibitors. We also screened a diverse library of molecules with unknown targets and identified chemical frameworks that show cardiotoxic signal in iPSC-CMs. By using this screening approach during target discovery and lead optimization, we can de-risk early-stage drug discovery. We show that the broad applicability of combining deep learning with iPSC technology is an effective way to interrogate cellular phenotypes and identify drugs that may protect against diseased phenotypes and deleterious mutations.

Originally Published in: eLife (2021) (Link to Paper)

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AI/ML PublicationsArianna KieserCardiomyocytes
High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing
High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing

Abstract: Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

Originally Published in: Sage Journals (2022) (Link to Paper)

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MantarrayArianna KieserSkeletal Muscle Cells
Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes
Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes

The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers’ response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.

Originally Published in: Nature Scientific Reports (2022) (Link to Paper)

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NautilusArianna KieserCardiomyocytes
Passive-Stretch Induced Skeletal Muscle Injury Platform for Duchenne Muscular Dystrophy Modeling

Inactivity following skeletal muscle dysfunction in DMD usually causes compromised soft tissue and decreased joint range of motion. Passive stretch techniques in combination with an exercise program are used as interventions to prevent musculoskeletal complications in children with DMD. However, the exact role of stretch-based rehabilitation methods is not well established in children with DMD. In fact, the underlying molecular and cellular mechanisms of how stretch-based rehabilitation methods in dystrophin-deficient muscle fibers might worsen the disease phenotype have not been fully explained. Therefore, the purpose was to establish an in vitro stretch-induced injury model in normal and dystrophic rat skeletal muscle fibers.

Originally Published in: Archives of Physical Medicine and Rehabilitation - V103, Issue 3 (2022) (Link to Paper)

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Curi CytostretcherArianna KieserSkeletal Muscle Cells
Using hiPSC-CMs to Examine Mechanisms of Catecholarminergic Polymorphic Ventricular Tachycardia

The integration of nanotopographic patterns with hiPSC-CM culture have been shown to induce structural and phenotypic development of hiPSC-CM monolayers by enhancing cytoskeletal organization and cellular alignment and providing greater sensitivity to conduction-blocking compounds and overall dose-response relationships (Carson et al., 2016; Smith et al., 2020). The NanoSurface platform by Curi Bio (Seattle, WA) can be used as a versatile platform for enhancing the structural development of cells and improving their physiological relevance in disease modeling. Culturing hiPSC-CMs on nanopatterned 25-mm coverslips (e.g., Curi Bio ANFSCS25) placed in a 6-well plate may facilitate the development of CPVT-specific cell morphology characteristics related to dyadic ultrastructure and contractile function through hiPSC-CM maturation.

Originally Published in: Current Protocols (2021) (Link to Paper)

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NanoSurface PlatesArianna KieserCPVT Phenotype hiPSC Model
Combining stretching and gallic acid to decrease inflammation indices and promote extracellular matrix production in osteoarthritic human articular chondrocytes

Osteoarthritis (OA) patients undergo cartilage degradation and experience painful joint swelling. OA symptoms are caused by inflammatory molecules and the upregulation of catabolic genes leading to the breakdown of cartilage extracellular matrix (ECM). Here, we investigate the effects of gallic acid (GA) and mechanical stretching on the expression of anabolic and catabolic genes and restoring ECM production by osteoarthritic human articular chondrocytes (hAChs) cultured in monolayers. hAChs were seeded onto conventional plates or silicone chambers with or without 100 μM GA. A 5% cyclic tensile strain (CTS) was applied to the silicone chambers and the deposition of collagen and glycosaminoglycan, and gene expressions of collagen types II (COL2A1), XI (COL11A2), I (COL1A1), and X (COL10A1), and matrix metalloproteinases (MMP-1 and MMP-13) as inflammation markers, were quantified. CTS and GA acted synergistically to promote the deposition of collagen and glycosaminoglycan in the ECM by 14- and 7-fold, respectively. Furthermore, the synergistic stimuli selectively upregulated the expression of cartilage-specific proteins, COL11A2 by 7-fold, and COL2A1 by 47-fold, and, in contrast, downregulated the expression of MMP-1 by 2.5-fold and MMP-13 by 125-fold. GA supplementation with CTS is a promising approach for restoring osteoarthritic hAChs ECM production ability making them suitable for complex tissue engineering applications.

Originally Published in: Experimental Cell Research - V408, Issue 2 (2021) (Link to Paper)

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Curi CytostretcherArianna KieserOsteoarthritic Human Articular Chondrocytes (hAChs)
BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization
BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization

Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis.

Originally Published in: International Journal of Molecular Sciences (2021) (Link to Paper)

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Curi CytostretcherArianna KieserHuman Endothelial Cells
Enhanced Matrix Production by Cocultivated Human Stem Cells and Chondrocytes Under Concurrent Mechanical Strain
Enhanced Matrix Production by Cocultivated Human Stem Cells and Chondrocytes Under Concurrent Mechanical Strain

A popular treatment for osteoarthritis is chondrocyte implantation. However, that treatment relies on cell survival and extracellular matrix production under high mechanical stress post-injection into patients' knees. In this study, adipose-derived stem cells and articular chondrocytes were cultured separately, or co-cultivated, at various ratios and exposed to mechanical stress to mimic forces that would be applied in patients' joints. This figure shows an increase in glycosaminoglycan (GAG) production which is an essential component of the extracellular matrix that lines articulating joints. Use of Curi Bio's Cytostretcher platform allowed researchers to identify the optimum culture conditions for cartilaginous ECM production under physiologically relevant conditions.

Originally Published in: In Vitro Cellular & Developmental Biology - Animal (2021) (Link to Paper)

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Curi CytostretcherArianna KieserStem Cells
Microtubules orchestrate local translation to enable cardiac growth

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation – and not simply translation rate – is a critical determinant of cardiac hypertrophy.

Originally Published in: Nature Communications (2021) (Link to Paper)

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NanoSurface PlatesArianna KieserNeonatal Rat Ventricular Myocytes (NRVMs)
Regional Differences and Physiologic Behaviors in Peripapillary Scleral Fibroblasts
Regional Differences and Physiologic Behaviors in Peripapillary Scleral Fibroblasts

Described cellular alignment in scleral stroma and showed that extracellular topography affects fibroblast activity in vitro. Specifically, demonstrated that extracellular topographic cues alter cellular response to mechanical strain and can stabilize fibroblasts by preventing a change in cellular orientation in the presence of mechanical strain.

Originally Published in: Investigative Ophthalmology and Visual Science (2021)

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Curi CytostretcherArianna Kieser
Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals
Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals

This study confirmed that focal adhesions transduce physical signals from topographical cues in the ECM into changes in cellular migration. Also, that aligned collagen organization induced myofibroblast differentiation, with a concomitant upregulation in myofibroblast-specific ECM genes like Postn and FnEDa.

Source: Darrian Bugg, Ross Bretherton, Peter Kim, Emily Olszewski, Abigail Nagle, Austin E. Schumacher, Nick Chu, Jagadambika Gunaje, Cole A. DeForest, Kelly Stevens, Deok-Ho Kim, Jennifer Davis. Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals. Circulation Research, October 2020 (Link to Paper)

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NanoSurface PlatesArianna KieserCardiac Tissue
A Live Cell Reporter of Exosome Secretion and Uptake Reveals Pathfinding Behavior of Migrating Cells
A Live Cell Reporter of Exosome Secretion and Uptake Reveals Pathfinding Behavior of Migrating Cells

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes and this study reports on a new fluorescent tool that allows for live imaging of cell migration. Researchers used this tool to visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Growing their cultures on nanopatterned plates allowed easy visualization of whether exosomes were secreted from the front or back of migrating cells, something impossible to determine on standard tissue culture plates. This new tool can now be used to understand a wide variety of roles for exosomes across tissue types.

Originally Published in: Nature Communications (2020) (Link to Paper)

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NanoSurface PlatesArianna Kieser
Cellular Locomotion Using Environmental Topography

This publication demonstrates that cells can transmit forces by coupling the retrograde flow of actin to a geometrically irregular environment, and that this can happen in the complete absence of any transmembrane receptors that link the cytoskeleton to the substrate.

Source: Reversat, A., Gaertner, F., Merrin, J. et al. Cellular locomotion using environmental topography. Nature 582, 582–585 (2020). (Link to Paper)

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NanoSurface PlatesArianna KieserT Cells
Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

This study demonstarted that the mechanical tension across cell surface proteins is responsive to changes in substrate stiffness, nanotopographical cues and to fluidic shear stresses. Moreover, it showed that these mechanical stimuli cause alterations in the association of cell surface proteins with cytoskeletal and focal adhesion-related signaling pathways.

Source: (Link to Paper)

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NanoSurface PlatesArianna KieserHuman Endothelial Cells