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Journal articleSchneider M, Fiedler L, Chapman K, et al., 2019,
MAP4K4 inhibition promotes survival of human stem cell derived cardiomyocyte and reduces infarct size in vivo
, Cell Stem Cell, Vol: 24, Pages: 579-591.e12, ISSN: 1875-9777Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.
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Conference paperWang BX, Kit-Anan W, Whittaker T, et al., 2019,
Human Cardiac Fibroblast-Secreted Exosomes Improve Efficiency of Human Cardiomyocyte Calcium Cycling
, Publisher: SPRINGER, Pages: 269-269, ISSN: 0920-3206 -
Journal articleArmstrong J, Maynard S, Pence I, et al., 2019,
Spatiotemporal quantification of acoustic cell patterning using Voronoi Tessellation
, Lab on a Chip, Vol: 19, Pages: 562-573, ISSN: 1473-0189Acoustic patterning using ultrasound standing waves has recently emerged as a potent biotechnology enabling the remote generation of ordered cell systems. This capability has opened up exciting opportunities, for example, in guiding the development of organoid cultures or the organization of complex tissues. The success of these studies is often contingent on the formation of tightly-packed and uniform cell arrays; however, a number of factors can act to disrupt or prevent acoustic patterning. Yet, to the best of our knowledge, there has been no comprehensive assessment of the quality of acoustically-patterned cell populations. In this report we use a mathematical approach, known as Voronoï tessellation, to generate a series of metrics that can be used to measure the effect of cell concentration, pressure amplitude, ultrasound frequency and biomaterial viscosity upon the quality of acoustically-patterned cell systems. Moreover, we extend this approach towards the characterization of spatiotemporal processes, namely, the acoustic patterning of cell suspensions and the migration of patterned, adherent cell clusters. This strategy is simple, unbiased and highly informative, and we anticipate that the methods described here will provide a systematic framework for all stages of acoustic patterning, including the robust quality control of devices, statistical comparison of patterning conditions, the quantitative exploration of parameter limits and the ability to track patterned tissue formation over time.
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Journal articleStejskalova A, Oliva Jorge N, England F, et al., 2019,
Biologically inspired, cell-selective release of aptamer-trapped growth factors by traction forces
, Advanced Materials, Vol: 31, Pages: 1-8, ISSN: 0935-9648Biomaterial scaffolds that are designed to incorporate dynamic, spatiotemporal information have the potential to interface with cells and tissues to direct behavior. Here we describe a bioinspired, programmable nanotechnology-based platform that harnesses cellular traction forces to activate growth factors, eliminating the need for exogenous triggers (e.g. light), spatially diffuse triggers (e.g. enzymes, pH changes) or passive activation (e.g. hydrolysis). We use flexible aptamer technology to create modular, synthetic mimics of the Large Latent Complex that restrains TGF-β1. This flexible nanotechnology-based approach is shown here to work with both platelet-derived growth factor-BB (PDGF-BB) and vascular endothelial growth factor (VEGF-165), integrate with glass coverslips, polyacrylamide gels, and collagen scaffolds, enable activation by various cells (e.g. primary human dermal fibroblasts, HMEC-1 endothelial cells) and unlock fundamentally new capabilities such as selective activation of growth factors by differing cell types (e.g. activation by smooth muscle cells but not fibroblasts) within clinically relevant collagen sponges.
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Journal articleDuarte D, Amarteifio S, Ang H, et al., 2019,
Defining the in vivo characteristics of acute myeloid leukemia cells behavior by intravital imaging
, Immunology and Cell Biology, Vol: 97, Pages: 229-235, ISSN: 0818-9641The majority of acute myeloid leukemia (AML) patients have a poor response to conventional chemotherapy. The survival of chemoresistant cells is thought to depend on leukemia-bone marrow (BM) microenvironment interactions, which are not well understood. The CXCL12/CXCR4 axis has been proposed to support AML growth but was not studied at the single AML cell level. We recently showed that T-cell acute lymphoblastic leukemia (T-ALL) cells are highly motile in the BM; however, the characteristics of AML cell migration within the BM remain undefined. Here, we characterize the in vivo migratory behavior of AML cells and their response to chemotherapy and CXCR4 antagonism, using high-resolution 2-photon and confocal intravital microscopy of mouse calvarium BM and the well-established MLL-AF9-driven AML mouse model. We used the Notch1-driven T-ALL model as a benchmark comparison and AMD3100 for CXCR4 antagonism experiments. We show that AML cells are migratory, and in contrast with T-ALL, chemoresistant AML cells become less motile. Moreover, and in contrast with T-ALL, the in vivo exploratory behavior of expanding and chemoresistant AML cells is unaffected by AMD3100. These results expand our understanding of AML cells-BM microenvironment interactions, highlighting unique traits of leukemia of different lineages.
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Journal articleSmith JGW, Owen T, Bhagwan JR, et al., 2018,
Isogenic pairs of hiPSC-CMs with hypertrophic cardiomyopathy/LVNC-associated ACTC1 E99K mutation unveil differential functional deficits
, Stem Cell Reports, Vol: 11, Pages: 1226-1243, ISSN: 2213-6711Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations.
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Journal articleDe Filippo K, Rankin SM, 2018,
CXCR4, the master regulator of neutrophil trafficking in homoeostasis and disease.
, European Journal of Clinical Investigation, Vol: 48, ISSN: 0014-2972BACKGROUND: Chemokines play a critical role in orchestrating the distribution and trafficking of neutrophils in homoeostasis and disease. RESULTS: The CXCR4/CXCL12 chemokine axis has been identified as a central regulator of these processes. CONCLUSION: In this review, we focus on the role of CXCR4/CXCL12 chemokine axis in regulating neutrophil release from the bone marrow and the trafficking of senescent neutrophils back to the bone marrow for clearance under homoeostasis and disease. We also discuss the role of CXCR4 in fine-tuning neutrophil responses in the context of inflammation.
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Journal articleArmstrong J, Puetzer JL, Serio A, et al., 2018,
Engineering anisotropic muscle tissue using acoustic cell patterning
, Advanced Materials, Vol: 30, Pages: 1-7, ISSN: 0935-9648Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.
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Journal articleLi C, Armstrong J, Pence I, et al., 2018,
Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering
, Biomaterials, Vol: 176, Pages: 24-33, ISSN: 0142-9612In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges.
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Journal articlePfanzelter J, Mostowy S, Way M, 2018,
Septins suppress the release of vaccinia virus from infected cells.
, J Cell Biol, Vol: 217, Pages: 2911-2929Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.
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