Publications by authors named "Christopher S Chen"

226 Publications

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

Am J Physiol Cell Physiol 2021 Dec 8. Epub 2021 Dec 8.

The Biological Design Center and Department of Biomedical Engineering, Boston University, Boston University, Boston, MA, United States.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.
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http://dx.doi.org/10.1152/ajpcell.00320.2021DOI Listing
December 2021

Directing Cholangiocyte Morphogenesis in Natural Biomaterial Scaffolds.

Adv Sci (Weinh) 2022 Jan 16;9(3):e2102698. Epub 2021 Nov 16.

Howard Hughes Medical Institute, Chevy Chase, MD, 20815, USA.

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.
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http://dx.doi.org/10.1002/advs.202102698DOI Listing
January 2022

Sarc-Graph: Automated segmentation, tracking, and analysis of sarcomeres in hiPSC-derived cardiomyocytes.

PLoS Comput Biol 2021 10 6;17(10):e1009443. Epub 2021 Oct 6.

Department of Mechanical Engineering, Boston University, Boston, Massachusetts, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pcbi.1009443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8523047PMC
October 2021

Probing the subcellular nanostructure of engineered human cardiomyocytes in 3D tissue.

Microsyst Nanoeng 2021 27;7:10. Epub 2021 Jan 27.

Department of Mechanical Engineering, Boston University, Boston, MA 02215 USA.

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 (  = 0.44) and lattice spacing (  = 0.46). The scattering intensity also correlates with lattice spacing (  = 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.
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http://dx.doi.org/10.1038/s41378-020-00234-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8433147PMC
January 2021

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

Circ Res 2021 09 18;129(7):751-766. Epub 2021 Aug 18.

Department of Genetics (R.A., C.N.T., Q.Z., J.G., S.R.D., C.E.S., J.G.S.), Harvard Medical School, Boston, MA.

[Figure: see text].
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317076DOI Listing
September 2021

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

J Virol 2021 09 9;95(19):e0086221. Epub 2021 Sep 9.

Department of Biochemistry, Boston Universitygrid.189504.1 School of Medicine, Boston, Massachusetts, USA.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.
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http://dx.doi.org/10.1128/JVI.00862-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428404PMC
September 2021

Direct laser writing for cardiac tissue engineering: a microfluidic heart on a chip with integrated transducers.

Lab Chip 2021 05;21(9):1724-1737

Department of Mechanical Engineering, Boston University, Boston, MA 02215, USA. and Photonics Center, Boston University, Boston, MA 02215, USA and Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA and Division of Materials Science and Engineering, Boston University, Boston, Massachusetts 02215, USA and Department of Physics, Boston University, Boston, MA 02215, USA.

We have developed a microfluidic platform for engineering cardiac microtissues in highly-controlled microenvironments. The platform is fabricated using direct laser writing (DLW) lithography and soft lithography, and contains four separate devices. Each individual device houses a cardiac microtissue and is equipped with an integrated strain actuator and a force sensor. Application of external pressure waves to the platform results in controllable time-dependent forces on the microtissues. Conversely, oscillatory forces generated by the microtissues are transduced into measurable electrical outputs. We demonstrate the capabilities of this platform by studying the response of cardiac microtissues derived from human induced pluripotent stem cells (hiPSC) under prescribed mechanical loading and pacing. This platform will be used for fundamental studies and drug screening on cardiac microtissues.
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http://dx.doi.org/10.1039/d0lc01078bDOI Listing
May 2021

Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair.

Nat Commun 2021 04 21;12(1):2359. Epub 2021 Apr 21.

Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.
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http://dx.doi.org/10.1038/s41467-021-22602-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060400PMC
April 2021

Fast, multiplane line-scan confocal microscopy using axially distributed slits.

Biomed Opt Express 2021 Mar 9;12(3):1339-1350. Epub 2021 Feb 9.

Department of Biomedical Engineering, Boston University, 44 Cummington Mall, Boston, MA 02215, USA.

The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.
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http://dx.doi.org/10.1364/BOE.417286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984773PMC
March 2021

Controlled Apoptosis of Stromal Cells to Engineer Human Microlivers.

Adv Funct Mater 2020 Nov 8;30(48). Epub 2020 Jun 8.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Engineered tissue models comprise a variety of multiplexed ensembles in which combinations of epithelial, stromal, and immune cells give rise to physiologic function. Engineering spatiotemporal control of cell-cell and cell-matrix interactions within these 3D multicellular tissues would represent a significant advance for tissue engineering. In this work, a new method, entitled CAMEO (Controlled Apoptosis in Multicellular tissues for Engineered Organogenesis) enables the non-invasive triggering of controlled apoptosis to eliminate genetically-engineered cells from a pre-established culture. Using this approach, the contribution of stromal cells to the phenotypic stability of primary human hepatocytes is examined. 3D hepatic microtissues, in which fibroblasts can enhance phenotypic stability and accelerate aggregation into spheroids, were found to rely only transiently on fibroblast interaction to support multiple axes of liver function, such as protein secretion and drug detoxification. Due to its modularity, CAMEO has the promise to be readily extendable to other applications that are tied to the complexity of 3D tissue biology, from understanding organoid models to building artificial tissue grafts.
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http://dx.doi.org/10.1002/adfm.201910442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996305PMC
November 2020

Controlled Cell Alignment Using Two-Photon Direct Laser Writing-Patterned Hydrogels in 2D and 3D.

Macromol Biosci 2021 05 19;21(5):e2100051. Epub 2021 Mar 19.

Department of Biomedical Engineering, Boston University, Boston, MA, 02215, USA.

Direct laser writing (DLW) via two-photon polymerization is an emerging highly precise technique for the fabrication of intricate cellular scaffolds. Despite recent progress in using two-photon-polymerized scaffolds to probe fundamental cell behaviors, new methods to direct and modulate microscale cell alignment and selective cell adhesion using two-photon-polymerized microstructures are of keen interest. Here, a DLW-fabricated 2D and 3D hydrogel microstructures, with alternating soft and stiff regions, for precisely controlled cell alignment are reported. The use of both cell-adhesive and cell-repellent hydrogels allows selective adhesion and alignment of human mesenchymal stem cells within the printed structure. Importantly, DLW patterning enables cell alignment on flat surfaces as well as irregular and curved 3D microstructures, which are otherwise challenging to pattern with cells.
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http://dx.doi.org/10.1002/mabi.202100051DOI Listing
May 2021

Optogenetic current in myofibroblasts acutely alters electrophysiology and conduction of co-cultured cardiomyocytes.

Sci Rep 2021 02 24;11(1):4430. Epub 2021 Feb 24.

Department of Biomedical Engineering, Johns Hopkins University, 720 Rutland Ave., Baltimore, MD, 21205, USA.

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 < 10), upstroke rate (- 13 ± 4%, p = 0.002), and action potential duration (- 14 ± 7%, p = 0.004) in co-cultures when 0.017 mW/mm light was applied, as well as focal spontaneous beating in 6/7 samples and a decreased cycle length (- 36 ± 18%, p = 0.002) at 0.057 mW/mm light. In silico modeling of the experiments reproduced the experimental findings and suggested the light levels used in experiments produced excess current similar in magnitude to endogenous myofibroblast current. Fitting the model to experimental data predicted a tissue-level electrical conductance across the 3-D interface between myofibroblasts and cardiomyocytes of ~ 5 nS/cardiomyocyte, and showed how increased myofibroblast-myocyte conductance, increased myofibroblast/myocyte capacitance ratio, and increased myofibroblast current, which occur in fibrosis, can work in tandem to produce pro-arrhythmic increases in conduction and spontaneous beating.
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http://dx.doi.org/10.1038/s41598-021-83398-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904933PMC
February 2021

Transient Support from Fibroblasts is Sufficient to Drive Functional Vascularization in Engineered Tissues.

Adv Funct Mater 2020 Nov 25;30(48). Epub 2020 Jun 25.

Biological Design Center, Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by co-culturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly could be useful for clinical translation. In this study, we introduce a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis), whereby fibroblasts are selectively ablated on demand, and utilize it to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, we demonstrate the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering-via-elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.
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http://dx.doi.org/10.1002/adfm.202003777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891457PMC
November 2020

Reconstituting the dynamics of endothelial cells and fibroblasts in wound closure.

APL Bioeng 2021 Mar 19;5(1):016102. Epub 2021 Jan 19.

The Biological Design Center and Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.

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 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.
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http://dx.doi.org/10.1063/5.0028651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817247PMC
March 2021

Harnessing Mechanobiology for Tissue Engineering.

Dev Cell 2021 01 15;56(2):180-191. Epub 2021 Jan 15.

Department of Biomedical Engineering and the Biological Design Center, Boston University, Boston, MA 02215, USA; The Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA. Electronic address:

A primary challenge in tissue engineering is to recapitulate both the structural and functional features of whole tissues and organs. In vivo, patterning of the body plan and constituent tissues emerges from the carefully orchestrated interactions between the transcriptional programs that give rise to cell types and the mechanical forces that drive the bending, twisting, and extensions critical to morphogenesis. Substantial recent progress in mechanobiology-understanding how mechanics regulate cell behaviors and what cellular machineries are responsible-raises the possibility that one can begin to use these insights to help guide the strategy and design of functional engineered tissues. In this perspective, we review and propose the development of different approaches, from providing appropriate extracellular mechanical cues to interfering with cellular mechanosensing machinery, to aid in controlling cell and tissue structure and function.
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http://dx.doi.org/10.1016/j.devcel.2020.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855912PMC
January 2021

Distinct effects of different matrix proteoglycans on collagen fibrillogenesis and cell-mediated collagen reorganization.

Sci Rep 2020 11 4;10(1):19065. Epub 2020 Nov 4.

Bioengineering Graduate Group, University of Pennsylvania, Philadelphia, PA, 19104, USA.

The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.
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http://dx.doi.org/10.1038/s41598-020-76107-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642422PMC
November 2020

SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway.

bioRxiv 2020 Oct 28. Epub 2020 Oct 28.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.
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http://dx.doi.org/10.1101/2020.10.27.358259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605551PMC
October 2020

REPLY.

Hepatology 2021 02;73(2):872-873

Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA.

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http://dx.doi.org/10.1002/hep.31471DOI Listing
February 2021

Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in and That Are Common in Chinese Patients.

Circ Genom Precis Med 2020 10 20;13(5):424-434. Epub 2020 Aug 20.

Cardiovascular Research Center, Royal Brompton Hospital, London, United Kingdom (N.W., A.d.M., R.G., R.J.B., P.J.R.B., J.S.W., S.A.C.).

Background: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry.

Methods: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies.

Results: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, <0.0001) but an excess of variants of uncertain significance (24%, <0.0001), as compared to Whites (pathogenic/likely pathogenic: 31%, excess of variants of uncertain significance: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency [AF]=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, =0.0057, genome aggregation database-East Asian AF=0.0062, =0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, <0.0001, genome aggregation database-East Asian AF=0.0009, <0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared with noncarriers while its etiological fraction is limited (0.70 [95% CI, 0.35-0.86]) and thus TNNI3:p.R79C is considered variant of uncertain significance. Mutant TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes) show hypercontractility, increased metabolic requirements, and cellular hypertrophy and the etiological fraction (0.93 [95% CI, 0.83-0.97]) support the likely pathogenicity of TNNT2:p.R286H.

Conclusions: As compared with Whites, Chinese HCM patients commonly have low penetrance risk alleles in or but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-White populations.
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http://dx.doi.org/10.1161/CIRCGEN.119.002823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676617PMC
October 2020

Uncovering mutation-specific morphogenic phenotypes and paracrine-mediated vessel dysfunction in a biomimetic vascularized mammary duct platform.

Nat Commun 2020 07 6;11(1):3377. Epub 2020 Jul 6.

Department of Biomedical Engineering, Boston University, 610 Commonwealth Ave, Boston, MA, 02215, USA.

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3Kα ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3Kα-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.
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http://dx.doi.org/10.1038/s41467-020-17102-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338408PMC
July 2020

Recovery of Tractions Exerted by Single Cells in Three-Dimensional Nonlinear Matrices.

J Biomech Eng 2020 08;142(8)

Department of Aerospace and Mechanical Engineering, University of Southern California, Los Angeles, CA 90089.

Cell-generated tractions play an important role in various physiological and pathological processes such as stem-cell differentiation, cell migration, wound healing, and cancer metastasis. Traction force microscopy (TFM) is a technique for quantifying cellular tractions during cell-matrix interactions. Most applications of this technique have heretofore assumed that the matrix surrounding the cells is linear elastic and undergoes infinitesimal strains, but recent experiments have shown that the traction-induced strains can be large (e.g., more than 50%). In this paper, we propose a novel three-dimensional (3D) TFM approach that consistently accounts for both the geometric nonlinearity introduced by large strains in the matrix, and the material nonlinearity due to strain-stiffening of the matrix. In particular, we pose the TFM problem as a nonlinear inverse hyperelasticity problem in the stressed configuration of the matrix, with the objective of determining the cellular tractions that are consistent with the measured displacement field in the matrix. We formulate the inverse problem as a constrained minimization problem and develop an efficient adjoint-based minimization procedure to solve it. We first validate our approach using simulated data, and quantify its sensitivity to noise. We then employ the new approach to recover tractions exerted by NIH 3T3 cells fully encapsulated in hydrogel matrices of varying stiffness. We find that neglecting nonlinear effects can induce significant errors in traction reconstructions. We also find that cellular tractions roughly increase with gel stiffness, while the strain energy appears to saturate.
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http://dx.doi.org/10.1115/1.4046974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477711PMC
August 2020

Voltage Imaging of Cardiac Cells and Tissue Using the Genetically Encoded Voltage Sensor Archon1.

iScience 2020 Apr 11;23(4):100974. Epub 2020 Mar 11.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.isci.2020.100974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160579PMC
April 2020

Mechanical regulation of glycolysis via cytoskeleton architecture.

Nature 2020 02 12;578(7796):621-626. Epub 2020 Feb 12.

Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility. Although all of these processes consume energy, it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.
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http://dx.doi.org/10.1038/s41586-020-1998-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210009PMC
February 2020

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

Circulation 2020 03 27;141(10):828-842. Epub 2020 Jan 27.

Department of Genetics, Harvard Medical School, Boston, MA (C.N.T., A.C.G., G.V., H.W., G.R., A.S., R.A., A.C.P., J.G.S., C.E.S.).

Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations.

Methods: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias.

Results: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM.

Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.042339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077965PMC
March 2020

From Simple to Architecturally Complex Hydrogel Scaffolds for Cell and Tissue Engineering Applications: Opportunities Presented by Two-Photon Polymerization.

Adv Healthc Mater 2020 01 20;9(1):e1901217. Epub 2019 Nov 20.

Department of Biomedical Engineering, Boston University, Boston, MA, 02215, USA.

Direct laser writing via two-photon polymerization (2PP) is an emerging micro- and nanofabrication technique to prepare predetermined and architecturally precise hydrogel scaffolds with high resolution and spatial complexity. As such, these scaffolds are increasingly being evaluated for cell and tissue engineering applications. This article first discusses the basic principles and photoresists employed in 2PP fabrication of hydrogels, followed by an in-depth introduction of various mechanical and biological characterization techniques used to assess the fabricated structures. The design requirements for cell and tissue related applications are then described to guide the engineering, physicochemical, and biological efforts. Three case studies in bone, cancer, and cardiac tissues are presented that illustrate the need for structured materials in the next generation of clinical applications. This paper concludes by summarizing the progress to date, identifying additional opportunities for 2PP hydrogel scaffolds, and discussing future directions for 2PP research.
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http://dx.doi.org/10.1002/adhm.201901217DOI Listing
January 2020

A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling.

Sci Adv 2019 08 28;5(8):eaav6789. Epub 2019 Aug 28.

Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.
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http://dx.doi.org/10.1126/sciadv.aav6789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713506PMC
August 2019

A Bile Duct-on-a-Chip With Organ-Level Functions.

Hepatology 2020 04 28;71(4):1350-1363. Epub 2019 Oct 28.

Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA.

Background And Aims: Chronic cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), are frequently associated with damage to the barrier function of the biliary epithelium. Here, we report on a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions.

Approach And Results: We showed that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, that the permeability of the cholangiocyte monolayer was comparable to ex vivo measurements, and that cholangiocytes in the device were mechanosensitive (as demonstrated by changes in calcium flux under applied luminal flow). Permeability decreased significantly when cells formed a compact monolayer with cell densities comparable to those observed in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin, biliatresone, and the bile acid, glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid.

Conclusions: This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents an in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.
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http://dx.doi.org/10.1002/hep.30918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048662PMC
April 2020

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues.

ACS Biomater Sci Eng 2019 Aug 20;5(8):3843-3855. Epub 2018 Dec 20.

Department of Physics and Astronomy, Johns Hopkins University, Baltimore, Maryland 21218, United States.

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues' contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues' Young's moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues' properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships.
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http://dx.doi.org/10.1021/acsbiomaterials.8b01183DOI Listing
August 2019
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