Publications by authors named "Gaspard Pardon"

11 Publications

  • Page 1 of 1

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Proc Natl Acad Sci U S A 2021 Jun;118(24)

Department of Pediatrics (Cardiology), Stanford University School of Medicine, Palo Alto, CA 94304;

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.
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http://dx.doi.org/10.1073/pnas.2025030118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214707PMC
June 2021

Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.

Stem Cell Reports 2021 May 11. Epub 2021 May 11.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CCSR Room 4215, 269 Campus Drive, Stanford, CA 94305-5175, USA; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.
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http://dx.doi.org/10.1016/j.stemcr.2021.04.018DOI Listing
May 2021

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies.

J Micromech Microeng 2019 Nov 26;29(11). Epub 2019 Sep 26.

Department of Bioengineering, Stanford University, Stanford, California.

Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.
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http://dx.doi.org/10.1088/1361-6439/ab419aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457726PMC
November 2019

Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

Cell Mol Bioeng 2020 Feb 25;13(1):87-98. Epub 2019 Sep 25.

Laboratory for Complex Fluids and Interfaces, Mechanobiology and Soft Matter Group, Research Institute for Biosciences, University of Mons, Place du Parc, 20, 7000 Mons, Belgium.

Introduction: The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires assays with standardized orientations of collagen fibers.

Methods: To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes.

Results: Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution.

Conclusion: We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable assay for studying cell contact guidance mechanisms.
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http://dx.doi.org/10.1007/s12195-019-00600-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981333PMC
February 2020

Reaction injection molding of hydrophilic-in-hydrophobic femtolitre-well arrays.

Microsyst Nanoeng 2019 3;5:25. Epub 2019 Jun 3.

1Department of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden.

Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally.
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http://dx.doi.org/10.1038/s41378-019-0065-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545322PMC
June 2019

Engineering hiPSC cardiomyocyte in vitro model systems for functional and structural assessment.

Prog Biophys Mol Biol 2019 07 20;144:3-15. Epub 2018 Dec 20.

Departments of Mechanical Engineering and Bioengineering, Stanford University, Stanford, CA, 94305, USA; Department of Mechanical Engineering, University of California at Santa Barbara, USA.

The study of human cardiomyopathies and the development and testing of new therapies has long been limited by the availability of appropriate in vitro model systems. Cardiomyocytes are highly specialized cells whose internal structure and contractile function are sensitive to the local microenvironment and the combination of mechanical and biochemical cues they receive. The complementary technologies of human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) and microphysiological systems (MPS) allow for precise control of the genetics and microenvironment of human cells in in vitro contexts. These combined systems also enable quantitative measurement of mechanical function and intracellular organization. This review describes relevant factors in the myocardium microenvironment that affect CM structure and mechanical function and demonstrates the application of several engineered microphysiological systems for studying development, disease, and drug discovery.
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http://dx.doi.org/10.1016/j.pbiomolbio.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919215PMC
July 2019

Big bottlenecks in cardiovascular tissue engineering.

Commun Biol 2018 21;1:199. Epub 2018 Nov 21.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, 94305, CA, USA.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges.
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http://dx.doi.org/10.1038/s42003-018-0202-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249300PMC
November 2018

Sampling and detection of airborne influenza virus towards point-of-care applications.

PLoS One 2017 28;12(3):e0174314. Epub 2017 Mar 28.

KTH Royal Institute of Technology, Department of Micro and Nanosystems, Stockholm, Sweden.

Airborne transmission of the influenza virus contributes significantly to the spread of this infectious pathogen, particularly over large distances when carried by aerosol droplets with long survival times. Efficient sampling of virus-loaded aerosol in combination with a low limit of detection of the collected virus could enable rapid and early detection of airborne influenza virus at the point-of-care setting. Here, we demonstrate a successful sampling and detection of airborne influenza virus using a system specifically developed for such applications. Our system consists of a custom-made electrostatic precipitation (ESP)-based bioaerosol sampler that is coupled with downstream quantitative polymerase chain reaction (qPCR) analysis. Aerosolized viruses are sampled directly into a miniaturized collector with liquid volume of 150 μL, which constitutes a simple and direct interface with subsequent biological assays. This approach reduces sample dilution by at least one order of magnitude when compared to other liquid-based aerosol bio-samplers. Performance of our ESP-based sampler was evaluated using influenza virus-loaded sub-micron aerosols generated from both cultured and clinical samples. Despite the miniaturized collection volume, we demonstrate a collection efficiency of at least 10% and sensitive detection of a minimum of 3721 RNA copies. Furthermore, we show that an improved extraction protocol can allow viral recovery of down to 303 RNA copies and a maximum sampler collection efficiency of 47%. A device with such a performance would reduce sampling times dramatically, from a few hours with current sampling methods down to a couple of minutes with our ESP-based bioaerosol sampler.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174314PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369763PMC
September 2017

Single-Step Imprinting of Femtoliter Microwell Arrays Allows Digital Bioassays with Attomolar Limit of Detection.

ACS Appl Mater Interfaces 2017 Mar 15;9(12):10418-10426. Epub 2017 Mar 15.

Department of Biosystems, KU Leuven-University of Leuven , Willem de Croylaan 42, 3001 Leuven, Belgium.

Bead-based microwell array technology is growing as an ultrasensitive analysis tool as exemplified by the successful commercial applications from Illumina and Quanterix for nucleic acid analysis and ultrasensitive protein measurements, respectively. High-efficiency seeding of magnetic beads is key for these applications and is enhanced by hydrophilic-in-hydrophobic microwell arrays, which are unfortunately often expensive or labor-intensive to manufacture. Here, we demonstrate a new single-step manufacturing approach for imprinting cheap and disposable hydrophilic-in-hydrophobic microwell arrays suitable for digital bioassays. Imprinting of arrays with hydrophilic-in-hydrophobic microwells is made possible using an innovative surface energy replication approach by means of a hydrophobic thiol-ene polymer formulation. In this polymer, hydrophobic-moiety-containing monomers self-assemble at the hydrophobic surface of the imprinting stamp, which results in a hydrophobic replica surface after polymerization. After removing the stamp, microwells with hydrophobic walls and a hydrophilic bottom are obtained. We demonstrate that the hydrophilic-in-hydrophobic imprinted microwell arrays enable successful and efficient self-assembly of individual water droplets and seeding of magnetic beads with loading efficiencies up to 96%. We also demonstrate the suitability of the microwell arrays for the isolation and digital counting of single molecules achieving a limit of detection of 17.4 aM when performing a streptavidin-biotin binding assay as model system. Since this approach is up-scalable through reaction injection molding, we expect it will contribute substantially to the translation of ultrasensitive digital microwell array technology toward diagnostic applications.
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http://dx.doi.org/10.1021/acsami.6b15415DOI Listing
March 2017

Off-stoichiometry improves the photostructuring of thiol-enes through diffusion-induced monomer depletion.

Microsyst Nanoeng 2016 15;2:15043. Epub 2016 Feb 15.

Micro and Nanosystems, KTH Royal Institute of Technology, Osquldas väg 10, SE-10044, Stockholm, Sweden.

Thiol-enes are a group of alternating copolymers with highly ordered networks and are used in a wide range of applications. Here, "click" chemistry photostructuring in off-stoichiometric thiol-enes is shown to induce microscale polymeric compositional gradients due to species diffusion between non-illuminated and illuminated regions, creating two narrow zones with distinct compositions on either side of the photomask feature boundary: a densely cross-linked zone in the illuminated region and a zone with an unpolymerized highly off-stoichiometric monomer composition in the non-illuminated region. Using confocal Raman microscopy, it is here explained how species diffusion causes such intricate compositional gradients in the polymer and how off-stoichiometry results in improved image transfer accuracy in thiol-ene photostructuring. Furthermore, increasing the functional group off-stoichiometry and decreasing the photomask feature size is shown to amplify the induced gradients, which potentially leads to a new methodology for microstructuring.
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http://dx.doi.org/10.1038/micronano.2015.43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444721PMC
February 2016

Pt-Al2O3 dual layer atomic layer deposition coating in high aspect ratio nanopores.

Nanotechnology 2013 Jan 5;24(1):015602. Epub 2012 Dec 5.

KTH Royal Institute of Technology, School of Electrical Engineering, Micro and Nanosystems, Osquldas Väg 10, SE-10044 Stockholm, Sweden.

Functional nanoporous materials are promising for a number of applications ranging from selective biofiltration to fuel cell electrodes. This work reports the functionalization of nanoporous membranes using atomic layer deposition (ALD). ALD is used to conformally deposit platinum (Pt) and aluminum oxide (Al(2)O(3)) on Pt in nanopores to form a metal-insulator stack inside the nanopore. Deposition of these materials inside nanopores allows the addition of extra functionalities to nanoporous materials such as anodic aluminum oxide (AAO) membranes. Conformal deposition of Pt on such materials enables increased performances for electrochemical sensing applications or fuel cell electrodes. An additional conformal Al(2)O(3) layer on such a Pt film forms a metal-insulator-electrolyte system, enabling field effect control of the nanofluidic properties of the membrane. This opens novel possibilities in electrically controlled biofiltration. In this work, the deposition of these two materials on AAO membranes is investigated theoretically and experimentally. Successful process parameters are proposed for a reliable and cost-effective conformal deposition on high aspect ratio three-dimensional nanostructures. A device consisting of a silicon chip supporting an AAO membrane of 6 mm diameter and 1.3 μm thickness with 80 nm diameter pores is fabricated. The pore diameter is reduced to 40 nm by a conformal deposition of 11 nm Pt and 9 nm Al(2)O(3) using ALD.
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http://dx.doi.org/10.1088/0957-4484/24/1/015602DOI Listing
January 2013
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