Publications by authors named "Charles E Murry"

179 Publications

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Cell Rep 2021 May;35(5):109088

Department of Genetics and Genome Sciences, UConn Health, Farmington, CT 06030, USA; The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA. Electronic address:

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.109088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8161465PMC
May 2021

Engrafted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Undergo Clonal Expansion In Vivo.

Circulation 2021 Apr 19;143(16):1635-1638. Epub 2021 Apr 19.

Department of Laboratory Medicine and Pathology (D.E.-N., D.B., A.M.M., C.E.M., N.J.S., J.D.), University of Washington, Seattle.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059012PMC
April 2021

Tunable electroconductive decellularized extracellular matrix hydrogels for engineering human cardiac microphysiological systems.

Biomaterials 2021 05 18;272:120764. Epub 2021 Mar 18.

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21205, USA; Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, 21205, USA. Electronic address:

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) offer tremendous potential when used to engineer human tissues for drug screening and disease modeling; however, phenotypic immaturity reduces assay reliability when translating in vitro results to clinical studies. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cell and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces and had increased expression of genes that regulate contractile function. Improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. dECM-rGO hydrogels could also be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced electrophysiological function of these tissues corresponded well with a capability to produce physiologically relevant drug responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2021.120764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074529PMC
May 2021

SARS-CoV-2 Infects Human Pluripotent Stem Cell-Derived Cardiomyocytes, Impairing Electrical and Mechanical Function.

Stem Cell Reports 2021 03 13;16(3):478-492. Epub 2021 Feb 13.

Department of Laboratory Medicine and Pathology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA; Center for Cardiovascular Biology, University of Washington, 850 Republican Street, Brotman Building, Seattle, WA 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, 850 Republican Street, Seattle, WA 98109, USA; Sana Biotechnology, 188 E Blaine Street, Seattle, WA 98102, USA; Department of Bioengineering, University of Washington, 3720 15th Avenue NE, Seattle, WA 98105, USA; Department of Medicine/Cardiology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA. Electronic address:

COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2021.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881699PMC
March 2021

Cell Therapy Strategies With No Safety Concerns and Demonstrated Benefits Warrant Study - Reply.

Circ J 2020 10 10;84(11):2122. Epub 2020 Oct 10.

Institute for Stem Cell and Regenerative Medicine, University of Washington.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1253/circj.CJ-20-0928DOI Listing
October 2020

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and Their Differentiated Derivatives.

Stem Cell Reports 2020 07 2;15(1):226-241. Epub 2020 Jul 2.

Department of Pathology, University of Washington, Seattle, WA 98109, USA; The Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA; Department of Bioengineering, University of Washington, Seattle, WA 98105, USA; Center for Cardiovascular Biology, University of Washington, Seattle, WA 98109, USA. Electronic address:

Single-cell transcriptomic approaches have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single-cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single-cell behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363961PMC
July 2020

Delta-1 Functionalized Hydrogel Promotes hESC-Cardiomyocyte Graft Proliferation and Maintains Heart Function Post-Injury.

Mol Ther Methods Clin Dev 2020 Jun 18;17:986-998. Epub 2020 Apr 18.

Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA.

Current cell transplantation techniques are hindered by small graft size, requiring high cell doses to achieve therapeutic cardiac remuscularization. Enhancing the proliferation of transplanted human embryonic stem cell-derived cardiomyocytes (hESC-CMs) could address this, allowing an otherwise subtherapeutic cell dose to prevent disease progression after myocardial infarction. In this study, we designed a hydrogel that activates Notch signaling through 3D presentation of the Notch ligand Delta-1 to use as an injectate for transplanting hESC-CMs into the infarcted rat myocardium. After 4 weeks, hESC-CM proliferation increased 2-fold and resulted in a 3-fold increase in graft size with the Delta-1 hydrogel compared to controls. To stringently test the effect of Notch-mediated graft expansion on long-term heart function, a normally subtherapeutic dose of hESC-CMs was implanted into the infarcted myocardium and cardiac function was evaluated by echocardiography. Transplantation of the Delta-1 hydrogel + hESC-CMs augmented heart function and was significantly higher at 3 months compared to controls. Graft size and hESC-CM proliferation were also increased at 3 months post-implantation. Collectively, these results demonstrate the therapeutic approach of a Delta-1 functionalized hydrogel to reduce the cell dose required to achieve functional benefit after myocardial infarction by enhancing hESC-CM graft size and proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2020.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225377PMC
June 2020

Engineering anisotropic 3D tubular tissues with flexible thermoresponsive nanofabricated substrates.

Biomaterials 2020 05 14;240:119856. Epub 2020 Feb 14.

Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA; Center for Cardiovascular Biology, University of Washington, Seattle, WA, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 20205, USA; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 20205, USA. Electronic address:

Tissue engineering aims to capture the structural and functional aspects of diverse tissue types in vitro. However, most approaches are limited in their ability to produce complex 3D geometries that are essential for tissue function. Tissues, such as the vasculature or chambers of the heart, often possess curved surfaces and hollow lumens that are difficult to recapitulate given their anisotropic architecture. Cell-sheet engineering techniques using thermoresponsive substrates provide a means to stack individual layers of cells with spatial control to create dense, scaffold-free tissues. In this study, we developed a novel method to fabricate complex 3D structures by layering multiple sheets of aligned cells onto flexible scaffolds and casting them into hollow tubular geometries using custom molds and gelatin hydrogels. To enable the fabrication of 3D tissues, we adapted our previously developed thermoresponsive nanopatterned cell-sheet technology by applying it to flexible substrates that could be folded as a form of tissue origami. We demonstrated the versatile nature of this platform by casting aligned sheets of smooth and cardiac muscle cells circumferentially around the surfaces of gelatin hydrogel tubes with hollow lumens. Additionally, we patterned skeletal muscle in the same fashion to recapitulate the 3D curvature that is observed in the muscles of the trunk. The circumferential cell patterning in each case was maintained after one week in culture and even encouraged organized skeletal myotube formation. Additionally, with the application of electrical field stimulation, skeletal myotubes began to assemble functional sarcomeres that could contract. Cardiac tubes could spontaneously contract and be paced for up to one month. Our flexible cell-sheet engineering approach provides an adaptable method to recapitulate more complex 3D geometries with tissue specific customization through the addition of different cell types, mold shapes, and hydrogels. By enabling the fabrication of scaled biomimetic models of human tissues, this approach could potentially be used to investigate tissue structure-function relationships, development, and maturation in the dish.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2020.119856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536133PMC
May 2020

Stem cells and the heart-the road ahead.

Science 2020 Feb;367(6480):854-855

Center for Cardiovascular Biology and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaz3650DOI Listing
February 2020

Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.

Nat Rev Cardiol 2020 06 3;17(6):341-359. Epub 2020 Feb 3.

Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA.

Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review, we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues with electromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41569-019-0331-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239749PMC
June 2020

Sonic Hedgehog upregulation does not enhance the survival and engraftment of stem cell-derived cardiomyocytes in infarcted hearts.

PLoS One 2020 16;15(1):e0227780. Epub 2020 Jan 16.

Department of Pathology, Center for Cardiovascular Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, United States of America.

The engraftment of human stem cell-derived cardiomyocytes (hSC-CMs) is a promising treatment for remuscularizing the heart wall post-infarction, but it is plagued by low survival of transplanted cells. We hypothesize that this low survival rate is due to continued ischemia within the infarct, and that increasing the vascularization of the scar will ameliorate the ischemia and improve hSC-CM survival and engraftment. An adenovirus expressing the vascular growth factor Sonic Hedgehog (Shh) was injected into the infarcted myocardium of rats immediately after ischemia/reperfusion, four days prior to hSC-CM injection. By two weeks post-cell injection, Shh treatment had successfully increased capillary density outside the scar, but not within the scar. In addition, there was no change in vessel size or percent vascular volume when compared to cell injection alone. Micro-computed tomography revealed that Shh failed to increase the number and size of larger vessels. It also had no effect on graft size or heart function when compared to cell engraftment alone. Our data suggests that, when combined with the engraftment of hSC-CMs, expression of Shh within the infarct scar and surrounding myocardium is unable to increase vascularization of the infarct scar, and it does not improve survival or function of hSC-CM grafts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227780PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964843PMC
April 2020

NanoMEA: A Tool for High-Throughput, Electrophysiological Phenotyping of Patterned Excitable Cells.

Nano Lett 2020 03 23;20(3):1561-1570. Epub 2019 Dec 23.

Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States.

Matrix nanotopographical cues are known to regulate the structure and function of somatic cells derived from human pluripotent stem cell (hPSC) sources. High-throughput electrophysiological analysis of excitable cells derived from hPSCs is possible via multielectrode arrays (MEAs) but conventional MEA platforms use flat substrates and do not reproduce physiologically relevant tissue-specific architecture. To address this issue, we developed a high-throughput nanotopographically patterned multielectrode array (nanoMEA) by integrating conductive, ion-permeable, nanotopographic patterns with 48-well MEA plates, and investigated the effect of substrate-mediated cytoskeletal organization on hPSC-derived cardiomyocyte and neuronal function at scale. Using our nanoMEA platform, we found patterned hPSC-derived cardiac monolayers exhibit both enhanced structural organization and greater sensitivity to treatment with calcium blocking or conduction inhibiting compounds when subjected to high-throughput dose-response studies. Similarly, hPSC-derived neurons grown on nanoMEA substrates exhibit faster migration and neurite outgrowth speeds, greater colocalization of pre- and postsynaptic markers, and enhanced cell-cell communication only revealed through examination of data sets derived from multiple technical replicates. The presented data highlight the nanoMEA as a new tool to facilitate high-throughput, electrophysiological analysis of ordered cardiac and neuronal monolayers, which can have important implications for preclinical analysis of excitable cell function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.nanolett.9b04152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547911PMC
March 2020

Function Follows Form - A Review of Cardiac Cell Therapy.

Circ J 2019 11 13;83(12):2399-2412. Epub 2019 Nov 13.

Institute for Stem Cell and Regenerative Medicine, University of Washington.

The investment of nearly 2 decades of clinical investigation into cardiac cell therapy has yet to change cardiovascular practice. Recent insights into the mechanism of cardiac regeneration help explain these results and provide important context in which we can develop next-generation therapies. Non-contractile cells such as bone marrow or adult heart derivatives neither engraft long-term nor induce new muscle formation. Correspondingly, these cells offer little functional benefit to infarct patients. In contrast, preclinical data indicate that transplantation of bona fide cardiomyocytes derived from pluripotent stem cells induces direct remuscularization. This new myocardium beats synchronously with the host heart and induces substantial contractile benefits in macaque monkeys, suggesting that regeneration of contractile myocardium is required to fully recover function. Through a review of the preclinical and clinical trials of cardiac cell therapy, distinguishing the primary mechanism of benefit as either contractile or non-contractile helps appreciate the barriers to cardiac repair and establishes a rational path to optimizing therapeutic benefit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1253/circj.CJ-19-0567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002039PMC
November 2019

A Proteomic Perspective on Cardiomyocyte Maturation.

Circ Res 2019 11 7;125(11):954-956. Epub 2019 Nov 7.

From the Institute for Stem Cell and Regenerative Medicine (N.M., C.E.M.), Department of Pathology, University of Washington, Seattle.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.119.316039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003684PMC
November 2019

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes.

Nat Commun 2019 10 11;10(1):4671. Epub 2019 Oct 11.

Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-12482-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789043PMC
October 2019

Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function.

Circulation 2019 11 7;140(20):1647-1660. Epub 2019 Oct 7.

Department of Bioengineering (R.J.Z., J.M., D.-H.K., M.R., C.E.M.), University of Washington, Seattle.

Background: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin () are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function.

Methods: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z) and A-band (TTN-A) regions of the gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements.

Results: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z-CMs had sarcomeres and visibly contracted, whereas TTN-A-CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z-CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z-CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin.

Conclusions: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.039521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911360PMC
November 2019

Fatty Acids Enhance the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells.

Stem Cell Reports 2019 10 26;13(4):657-668. Epub 2019 Sep 26.

Department of Pathology, University of Washington, Seattle, WA 98109, USA; Department of Bioengineering, University of Washington, Seattle, WA 98109, USA; Department of Medicine/Cardiology, University of Washington, Seattle, WA 98109, USA; Center for Cardiovascular Biology, University of Washington, Seattle, WA 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, 850 Republican Street, Brotman Building Room 453, Seattle, WA 98109, USA. Electronic address:

Although human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a novel platform for heart regeneration, disease modeling, and drug screening, their immaturity significantly hinders their application. A hallmark of postnatal cardiomyocyte maturation is the metabolic substrate switch from glucose to fatty acids. We hypothesized that fatty acid supplementation would enhance hPSC-CM maturation. Fatty acid treatment induces cardiomyocyte hypertrophy and significantly increases cardiomyocyte force production. The improvement in force generation is accompanied by enhanced calcium transient peak height and kinetics, and by increased action potential upstroke velocity and membrane capacitance. Fatty acids also enhance mitochondrial respiratory reserve capacity. RNA sequencing showed that fatty acid treatment upregulates genes involved in fatty acid β-oxidation and downregulates genes in lipid synthesis. Signal pathway analyses reveal that fatty acid treatment results in phosphorylation and activation of multiple intracellular kinases. Thus, fatty acids increase human cardiomyocyte hypertrophy, force generation, calcium dynamics, action potential upstroke velocity, and oxidative capacity. This enhanced maturation should facilitate hPSC-CM usage for cell therapy, disease modeling, and drug/toxicity screens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2019.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829750PMC
October 2019

Substrate Stiffness, Cell Anisotropy, and Cell-Cell Contact Contribute to Enhanced Structural and Calcium Handling Properties of Human Embryonic Stem Cell-Derived Cardiomyocytes.

ACS Biomater Sci Eng 2019 Aug 20;5(8):3876-3888. Epub 2019 Feb 20.

Department of Mechanical Engineering, University of Washington, Seattle, Washington 98195, United States.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can be utilized to understand the mechanisms underlying the development and progression of heart disease, as well as to develop better interventions and treatments for this disease. However, these cells are structurally and functionally immature, which undermines some of their adequacy in modeling adult heart tissue. Previous studies with immature cardiomyocytes have shown that altering substrate stiffness, cell anisotropy, and/or cell-cell contact can enhance the contractile and structural maturation of hPSC-CMs. In this study, the structural and calcium handling properties of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were enhanced by exposure to a downselected combination of these three maturation stimuli. First, hESC-CMs were seeded onto substrates composed of two commercial formulations of polydimethylsiloxane (PDMS), Sylgard 184 and Sylgard 527, whose stiffness ranged from 5 kPa to 101 kPa. Upon analyzing the morphological and calcium transient properties of these cells, it was concluded that a 21 kPa substrate yielded cells with the highest degree of maturation. Next, these PDMS substrates were microcontact-printed with laminin to force the cultured cells into rod-shaped geometries using line patterns that were 12, 18, or 24 μm in width. We found that cells on the 18 and 24 μm pattern widths had structural and functional properties that were superior to those on the 12 μm pattern. The hESC-CMs were then seeded onto these line-stamped surfaces at a density of 500 000 cells per 25-mm-diameter substrate, to enable the formation of cell-cell contacts at their distal ends. We discovered that this combination of culture conditions resulted in cells that were more structurally and functionally mature than those that were only exposed to one or two stimuli. Our results suggest that downselecting a combination of mechanobiological stimuli could prove to be an effective means of maturing hPSC-CMs in vitro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsbiomaterials.8b01256DOI Listing
August 2019

Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy.

J Cell Biol 2019 09 8;218(9):2919-2944. Epub 2019 Aug 8.

Department of Pathology, University of Washington, Seattle, WA

Mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared with gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape segregation to the nuclear lamina and inactivation during cardiogenesis. These regions exhibit up-regulation of multiple noncardiac genes including , encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. However, chromatin compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. Thus, global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201902117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719452PMC
September 2019

Learn from Your Elders: Developmental Biology Lessons to Guide Maturation of Stem Cell-Derived Cardiomyocytes.

Pediatr Cardiol 2019 Oct 6;40(7):1367-1387. Epub 2019 Aug 6.

Department of Pathology, University of Washington, 1959 NE Pacific Street, Seattle, WA, 98195, USA.

Human pluripotent stem cells (hPSCs) offer a multifaceted platform to study cardiac developmental biology, understand disease mechanisms, and develop novel therapies. Remarkable progress over the last two decades has led to methods to obtain highly pure hPSC-derived cardiomyocytes (hPSC-CMs) with reasonable ease and scalability. Nevertheless, a major bottleneck for the translational application of hPSC-CMs is their immature phenotype, resembling that of early fetal cardiomyocytes. Overall, bona fide maturation of hPSC-CMs represents one of the most significant goals facing the field today. Developmental biology studies have been pivotal in understanding the mechanisms to differentiate hPSC-CMs. Similarly, evaluation of developmental cues such as electrical and mechanical activities or neurohormonal and metabolic stimulations revealed the importance of these pathways in cardiomyocyte physiological maturation. Those signals cooperate and dictate the size and the performance of the developing heart. Likewise, this orchestra of stimuli is important in promoting hPSC-CM maturation, as demonstrated by current in vitro maturation approaches. Different shades of adult-like phenotype are achieved by prolonging the time in culture, electromechanical stimulation, patterned substrates, microRNA manipulation, neurohormonal or metabolic stimulation, and generation of human-engineered heart tissue (hEHT). However, mirroring this extremely dynamic environment is challenging, and reproducibility and scalability of these approaches represent the major obstacles for an efficient production of mature hPSC-CMs. For this reason, understanding the pattern behind the mechanisms elicited during the late gestational and early postnatal stages not only will provide new insights into postnatal development but also potentially offer new scalable and efficient approaches to mature hPSC-CMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00246-019-02165-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786957PMC
October 2019

Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration.

Nat Biotechnol 2019 08 2;37(8):895-906. Epub 2019 Aug 2.

The Anne McLaren Laboratory, Wellcome Trust - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.

The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived epicardium to augment the structure and function of engineered heart tissue in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells markedly enhanced the contractility, myofibril structure and calcium handling of human engineered heart tissues, while reducing passive stiffness compared with mesenchymal stromal cells. Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Cotransplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, cotransplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function makes them a promising adjuvant therapeutic for cardiac repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-019-0197-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824587PMC
August 2019

The K219T-Lamin mutation induces conduction defects through epigenetic inhibition of SCN5A in human cardiac laminopathy.

Nat Commun 2019 05 22;10(1):2267. Epub 2019 May 22.

Institute of Genetic and Biomedical Research (IRGB), UOS of Milan, National Research Council of Italy, Milan, 20138, Italy.

Mutations in LMNA, which encodes the nuclear proteins Lamin A/C, can cause cardiomyopathy and conduction disorders. Here, we employ induced pluripotent stem cells (iPSCs) generated from human cells carrying heterozygous K219T mutation on LMNA to develop a disease model. Cardiomyocytes differentiated from these iPSCs, and which thus carry K219T-LMNA, have altered action potential, reduced peak sodium current and diminished conduction velocity. Moreover, they have significantly downregulated Na1.5 channel expression and increased binding of Lamin A/C to the promoter of SCN5A, the channel's gene. Coherently, binding of the Polycomb Repressive Complex 2 (PRC2) protein SUZ12 and deposition of the repressive histone mark H3K27me3 are increased at SCN5A. CRISPR/Cas9-mediated correction of the mutation re-establishes sodium current density and SCN5A expression. Thus, K219T-LMNA cooperates with PRC2 in downregulating SCN5A, leading to decreased sodium current density and slower conduction velocity. This mechanism may underlie the conduction abnormalities associated with LMNA-cardiomyopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09929-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531493PMC
May 2019

Absence of full-length dystrophin impairs normal maturation and contraction of cardiomyocytes derived from human-induced pluripotent stem cells.

Cardiovasc Res 2020 02;116(2):368-382

Bioengineering, University of Washington, Seattle, WA, USA.

Aims: Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient.

Methods And Results: The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG-CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80-100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils.

Conclusions: Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvz109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053310PMC
February 2020

Lost in the fire.

Science 2019 04;364(6436):123-124

Department of Pathology, University of Washington, 850 Republican Street, Seattle, WA 98109, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aax1006DOI Listing
April 2019

Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory.

Nat Commun 2019 04 4;10(1):1538. Epub 2019 Apr 4.

Department of Pathology, University of Washington, 1959 NE Pacific Street, Seattle, WA, 98195, USA.

Functional changes in spatial genome organization during human development are poorly understood. Here we report a comprehensive profile of nuclear dynamics during human cardiogenesis from pluripotent stem cells by integrating Hi-C, RNA-seq and ATAC-seq. While chromatin accessibility and gene expression show complex on/off dynamics, large-scale genome architecture changes are mostly unidirectional. Many large cardiac genes transition from a repressive to an active compartment during differentiation, coincident with upregulation. We identify a network of such gene loci that increase their association inter-chromosomally, and are targets of the muscle-specific splicing factor RBM20. Genome editing studies show that TTN pre-mRNA, the main RBM20-regulated transcript in the heart, nucleates RBM20 foci that drive spatial proximity between the TTN locus and other inter-chromosomal RBM20 targets such as CACNA1C and CAMK2D. This mechanism promotes RBM20-dependent alternative splicing of the resulting transcripts, indicating the existence of a cardiac-specific trans-interacting chromatin domain (TID) functioning as a splicing factory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09483-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449405PMC
April 2019

Regenerating the field of cardiovascular cell therapy.

Nat Biotechnol 2019 03 18;37(3):232-237. Epub 2019 Feb 18.

Institute for Stem Cell Biology and Regenerative Medicine; Ludwig Center for Cancer Stem Cell Biology and Medicine; Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA.

The retraction of >30 falsified studies by Anversa et al. has had a disheartening impact on the cardiac cell therapeutics field. The premise of heart muscle regeneration by the transdifferentiation of bone marrow cells or putative adult resident cardiac progenitors has been largely disproven. Over the past 18 years, a generation of physicians and scientists has lost years chasing these studies, and patients have been placed at risk with little scientific grounding. Funding agencies invested hundreds of millions of dollars in irreproducible work, and both academic institutions and the scientific community ignored troubling signals over a decade of questionable work. Our collective retrospective analysis identifies preventable problems at the level of the editorial and peer-review process, funding agencies and academic institutions. This Perspective provides a chronology of the forces that led to this scientific debacle, integrating direct knowledge of the process. We suggest a science-driven path forward that includes multiple novel approaches to the problem of heart muscle regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-019-0042-1DOI Listing
March 2019

Patterned human microvascular grafts enable rapid vascularization and increase perfusion in infarcted rat hearts.

Nat Commun 2019 02 4;10(1):584. Epub 2019 Feb 4.

Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA.

Vascularization and efficient perfusion are long-standing challenges in cardiac tissue engineering. Here we report engineered perfusable microvascular constructs, wherein human embryonic stem cell-derived endothelial cells (hESC-ECs) are seeded both into patterned microchannels and the surrounding collagen matrix. In vitro, the hESC-ECs lining the luminal walls readily sprout and anastomose with de novo-formed endothelial tubes in the matrix under flow. When implanted on infarcted rat hearts, the perfusable microvessel grafts integrate with coronary vasculature to a greater degree than non-perfusable self-assembled constructs at 5 days post-implantation. Optical microangiography imaging reveal that perfusable grafts have 6-fold greater vascular density, 2.5-fold higher vascular velocities and >20-fold higher volumetric perfusion rates. Implantation of perfusable grafts containing additional hESC-derived cardiomyocytes show higher cardiomyocyte and vascular density. Thus, pre-patterned vascular networks enhance vascular remodeling and accelerate coronary perfusion, potentially supporting cardiac tissues after implantation. These findings should facilitate the next generation of cardiac tissue engineering design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-08388-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362250PMC
February 2019

Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells.

JACC Basic Transl Sci 2018 Dec 31;3(6):728-740. Epub 2018 Dec 31.

Department of Medicine/Cardiology, University of Washington, Seattle, Washington.

A novel myosin heavy chain 7 mutation (E848G) identified in a familial cardiomyopathy was studied in patient-specific induced pluripotent stem cell-derived cardiomyocytes. The cardiomyopathic human induced pluripotent stem cell-derived cardiomyocytes exhibited reduced contractile function as single cells and engineered heart tissues, and genome-edited isogenic cells confirmed the pathogenic nature of the E848G mutation. Reduced contractility may result from impaired interaction between myosin heavy chain 7 and cardiac myosin binding protein C.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacbts.2018.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314962PMC
December 2018