Additional Ventures’ multi-part virtual seminar series tackles diverse topics within and adjacent to the single ventricle field through the lens of investigators.

The Speaker Series began as a small gathering of single ventricle-centric researchers and clinicians participating in biweekly ‘Lunch & Learn’ sessions. It quickly grew into an interactive meeting of over 130 multidisciplinary investigators committed to advancing our understanding of single ventricle heart disease. 

The Details

Join your colleagues for a virtual seminar series where we feature three topic-specific sessions throughout the 2021-2022 academic year, Fall, Winter, and Spring. Each session is a four-week miniseries exploring the early emergence of the field and the need for cross-functional collaboration across the scientific spectrum. 

When: Join us every other Wednesday at 12 PM ET/ 9 AM PT beginning January 12, 2022.

Session format: A one-hour Zoom seminar with presentations from 2-3 investigators and plenty of time for discussion — our seminars are lively and interactive! Week 4 of each session features lightning round talks from 8-10 selected students, postdocs, and fellows.

Who’s invited: This series is open to all individuals from academic, clinical, nonprofit, or government entities. Please encourage your lab members, collaborators, and colleagues to attend!

How to participate: Please register to attend. You must register with a .edu, .gov, or .org email address.

 

Upcoming Session: Computational, Cellular, and Animal Models of Single Ventricle
The Winter Session features investigators at the leading edge of cardiovascular disease modeling. Each session will highlight perspectives from investigators using computational, cellular, and animal models – with an overarching goal to stimulate discussion and collaboration across fields.
Date
Topic
01/12/22

Future of Single Ventricle: Computational, Cellular, and Animal Models

The Congenital Heart Disease Cardiac Atlas Project

Andrew D. McCulloch, PhD, Shu Chien Chancellor’s Endowed Chair in Engineering and Medicine at the University of California San Diego and Director of the Institute for Engineering in Medicine

Abstract: The Cardiac Atlas Project uses computational modeling and machine learning to quantify and understand the wealth of structural and functional information available in cardiac magnetic resonance (CMR) imaging exams. By building statistical atlases that characterize the variation across populations and patient cohorts we are discovering new anatomical and functional features in CMR exams from adults and children with congenital heart diseases including tricuspid atresia and hypertrophic left heart syndrome with Fontan physiology and tetralogy of Fallot. By using mechanistic multiscale computational models, we can predict how structural defects contribute to functional impairment and arrhythmia risk. 

Mechanoregulation of fetal ventricular growth and maturation: emergence & complexity

Jonathan T. Butcher, PhD, Professor and Associate Director of the Nancy E. and Peter C. Meinig School of Biomedical Engineering at Cornell University

Abstract: Proper ventricular morphogenesis in humans requires the creation of bilaterally symmetric muscular pumping chambers, with trabeculated and compact domains, the latter with embedded conduction and vascular networks. Precise location and sizing of these domains is critical for gestational progression as each contributes vitally to the mechanical performance of the ensemble while sustaining further growth and maturation. While much is understood about mechanisms of early cardiogenesis, it is defects in later stages of growth and maturation that result in clinically serious malformations. While genetic manipulation has generated considerable insight into the contributions of individual cell lineages into cardiac formation, recent evidence elevates the potential that conditional signaling drives later growth and maturation events, in particular through sensation and response to their local hemodynamic environment. Our research group has developed novel experimental, imaging, and computational simulation technology to quantitatively monitor and perturb the environment of the fetal heart, using the chick animal model system. We have identified novel mechanically operated molecular switches that potentiate between growth and maturation motifs. Further, employing high resolution single cell and spatial RNA sequencing, we have further uncovered marked spatiotemporal differences in local cellular phenotype composition and signaling programs, which support local cellular composition as a previously unrecognized regulatory strategy for achieving structural heterogeneity. We here present some evidence towards this hypothesis as it relates to our efforts to understand and counteract mechanisms of ventricular malformation. 

 

01/26/22

Pioneers in Modeling Cardiovascular Disease

Development of a mouse model for investigating the cardiopulmonary remodeling after the Glenn Procedure

Tai Yi, MD, Microsurgery Center Director, Nationwide Children’s Hospital

Abstract: The Glenn shunt is a surgical procedure in which the superior vena cava is anastomosed to the pulmonary artery. The Glenn procedure is the second in a series of operations resulting in the creation of the Fontan circulation in which the venous circulation is directly connected to the pulmonary artery bypassing the right ventricle. This series of staged operations represent the current standard of care for patients born with single ventricle anomalies. While life saving, the Fontan procedure is associated with decreased longevity and significant life-long morbidity. The etiology underlying the multi-organ failure associated with Fontan circulation is poorly understood but is thought to be multifactorial arising from both the genetic factors associated with the underlying single ventricle disease in addition to the pathophysiological effects of Fontan hemodynamics. Mouse models provide a powerful tool for investigating the cellular and molecular mechanisms underlying disease. Herein we describe our initial results developing a mouse model for investigating the pathophysiological mechanisms underlying the Fontan circulation beginning with cardiopulmonary remodeling after the Glenn shunt.

Modeling collateral blood flow in regenerating and failing hearts

Kristy Red-Horse, PhD, Associate Professor of Biology at Stanford University and Howard Hughes Medical Institute Investigator

Abstract: Developing organisms create tissues de novo, and the underlying instructions could inform organ regeneration. With this mindset, we study coronary arteries—which bring blood flow to heart muscle—in hopes of eventually treating coronary artery disease, the number one killer worldwide. We have discovered how mouse coronary arteries are built, and reinstated developmental pathways in adults to aid recovery following cardiac injury.  

Generating the diversity of cardiovascular cell types from human pluripotent stem cells

Nicole Dubois, PhD, Associate Professor of Cell, Developmental and Regenerative Biology at the Icahn School of Medicine at Mount Sinai

Abstract: Many excellent strategies have been developed over the past years to generate the diversity of cardiovascular cell types in vitro from human pluripotent stem cells (hPSCs). However, there remain multiple open questions, including how to best mature in vitro-derived cells, how to generate complex tissue models or how to derive cells of the ventricular conduction system, for example. We show that transient Notch activation in ventricular cardiomyocytes results in stable induction of a Purkinje Fiber-like fate, including expression of conduction system markers, increased conduction velocity and adoption of Purkinje Fiber-like cell morphology. We further interrogated the role of metabolism for cardiac maturation, and have found that activation of PPAR signaling, in an isoform-specific manner, results in metabolic maturation of hPSC-derived cardiomyocytes, including enhanced fatty acid oxidation (FAO), expression of the FAO machinery, maturation of the mitochondrial network and enhanced sarcomere organization. Integrating these new strategies with existing approaches will enable the generation of relevant in vitro human models to study human heart development and disease.

02/09/22

Next Generation: Modeling Single Ventricle Heart Disease

Transcriptional and morphogenetic signatures of congenital heart disease pathways

Alessandro Bertero, PhD, Associate Professor in the Department of Molecular Biotechnology and Health Sciences at the University of Turin

Abstract: Approximately half of the patients with congenital heart disease (CHD) carry potentially damaging genetic variants. Yet in most cases, a definitive association for a given mutation to disease is lacking, as is an understanding of the cell type(s) affected and the underlying mechanism. These aspects greatly limit the usefulness of genetic testing as well as the development of targeted therapies. There is, therefore, a critical need to functionally annotate the role of a growing list of candidate CHD genes. Developing and validating rapid, scalable, and predictive models of CHD is equally key. We are pursuing these goals using human pluripotent stem cell (hiPSC)-derived cardiac progenitors and their derivatives. First, we have developed a new, phenotype-agnostic pooled screening approach called OPTiKD-seq (optimized inducible knockdown deconvoluted by sequencing). This method uses single-cell RNA-sequencing (scRNA-seq) to deconvolute the global effects of barcoded loss-of-function perturbations in hPSC-derived cells. Secondly, we are studying novel self-organizing hiPSC-derived cardiac organoids (cardioids) that form endothelial cells-lined chambered models of the first or second heart field. By comparing transcriptional and morphogenic signatures induced by CHD perturbations, we aim to advance genetic testing and provide the basis for the rational development and testing of personalized CHD therapies. 

Understanding Cardiac Malformations Through Targeted Cardiac Outflow Tract Mechanical Perturbation

Stephanie Lindsey, PhD,  Assistant Professor in the Mechanical and Aerospace Engineering Department at the University of California San Diego

Abstract: Hemodynamics plays a vital role in early cardiac morphogenesis. Disruption of established flow patterns during critical windows of development produces a range of defects that drastically alter function of the mature heart. Malformations of the outflow tract account for over 50% of clinically relevant congenital heart defects, yet the origin of such defects remains uncertain. A major limitation in determining causality of clinically relevant cardiac abnormalities is the difficulty of studying the effects of aberrant hemodynamics alone. Here, we use a combined experimental-computational approach to study the effects of altered flow dynamics on pre-programmed great vessel morphogenesis. Through the use of targeted nonlinear optics, we nucleate and control the growth of microbubbles within outflow vessels without disturbing surrounding tissues.  These targeted ablation experiments are coupled with subject-specific multiscale computational fluid dynamic models in order to track force propagation. Pressure, flow and wall shear maps obtained from these models serve as a basis for examining flow-mediated growth and adaptation in the heart and surrounding vessels. Results support a role for early great vessel hemodynamics in cardiac outflow tract rotation with wall shear stress presenting as a critical value to maintain. 

Models to Understand Vascular Adaptation in Single Ventricle Heart Disease

Abhay Ramachandra, PhD, Postdoctoral Associate in Biomedical Engineering (Jay Humphrey Lab) at Yale University

Abstract: Rewiring the circulation in single ventricle palliation surgeries subject the greater thoracic vessels to an altered hemodynamic environment. Not much is known about the ensuing longitudinal vascular remodeling, including adaptation versus maladaptation. Animal models provide the advantage of isolating the effects of individual insults, probing and gaining insights into these (mal)adaptations at multiple biological scales. I will present results of vascular remodeling in response to two insults to the circulatory systems in murine modelshypoxic injury and a Glenn surgery – both relevant to single ventricle heart disease. I will elaborate on the evolution of tissue mechanics from these perturbations and some associated changes in microstructure and cell signaling.  Finally, I will discuss how a multimodal data collection pipeline can inform computational models of vascular adaptation across biological scales and eventually lead to an integrative approach to guide vascular adaptations in single ventricle surgeries.

02/23/22

Lightning Round: Breaking Science from Students, Postdocs, and Fellows

Roster TBA

Interested in being featured as one of our Lightning Round speakers? See our Call for Abstracts!

We're sorry, there are no results for your query. Please try again.

Past Series

Date
Topic
10/20/21

Lightning Round: Breaking Science in Cardiac Tissue Engineering & Stem Cells

Do endothelial cells promote electrical maturation of stem cell-derived cardiomyocytes?

Jessica Garbern, MD, PhD, Postdoctoral Fellow, Harvard University, Department of Stem Cell and Regenerative Biology and Pediatric Cardiologist, Boston Children’s Hospital

Abstract: Clinical translation of stem cell therapies for heart disease is limited by a risk of potentially life-threatening ventricular arrhythmias seen following cardiomyocyte delivery in large animal models. Enhancing cardiomyocyte maturation may reduce this arrhythmogenic risk by reducing automaticity of delivered cardiomyocytes. We tested whether human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can enhance maturation and suppress automaticity of iPSC-derived cardiomyocytes (iPSC-CMs) in vitro. We found that co-culture of iPSC-ECs with iPSC-CMs significantly increased protein expression of cardiac troponin T, cardiac troponin I, Kir2.1, connexin 43, and CD36. In addition, using a stretchable mesh nanoelectronics device, we found that iPSC-ECs accelerated electrical maturation of iPSC-CMs. Using single cell RNA-seq, we identified differentially expressed surface markers that may facilitate purification of more mature iPSC-CMs. Further work will investigate whether these surface markers can identify and enrich for more mature iPSC-CM phenotypes.

Automated Vascular Design and Simulation for 3D Bioprinting

Zachary A. Sexton, Graduate Student at Stanford University Department of Bioengineering

Abstract: Incorporating perfusable vascular networks within engineered tissues and organs remains a grand challenge and precludes advancement towards artificial tissues at clinically relevant scales. Recent techniques in 3D biofabrication demonstrate the ability to manipulate living matter at resolutions and speeds necessary for viable tissues. However, there are currently no comprehensive algorithms to generate, model, and simulate biomimetic vascular networks for tissues of varying shape, vascular complexity, and perfusion conditions. Without a unified approach, efforts to vascularize tissues rely on simplified perfusion networks which poorly recapitulate pressure and flow distributions of native vasculature. We present a unified pipeline to meet this need through the open-source software package, SimVascular. This approach extends previous methods in constrained constructive optimization by partially binding local optimization routines and triaging collision repair to ensure efficient, accurate vascular generation. Further, new methods for implicit surface reconstruction are presented to efficiently vascularize nonconvex tissue and organ shapes with and without cavities. Thus, computational efficiency is maintained while perfusing more complex, biologic shapes. Resulting vascular networks can be exported for various 3D printing techniques as well as multiscale hemodynamic simulation. We demonstrate this comprehensive pipeline on a model of the ventricles to emphasize the application in whole tissue and organ vascular design planning. Additionally, model networks are presented for engineered shapes to meet desired bioreactor perfusion specifications and printing constraints.

Single Cell Multiomics Reveals Disrupted Gene Regulatory Networks in Congenital Heart Disease

Sanjeev Ranade, PhD, Postdoctoral Fellow at Gladstone Institute of Cardiovascular Disease

Abstract: A central question in developmental biology is understanding how disrupted gene regulatory networks in progenitor cells alter normal development and ultimately lead to disease. Spatiotemporal control of gene expression is essential for cell fate decisions and is coordinated in part by interactions between transcription factors (TFs) and cis regulatory elements (cREs) such as enhancers. Loss of function variants in TFs that lead to dysregulated gene expression patterns in development have been identified in patients with congenital heart defects (CHD). However, the disrupted activity of cREs within specific cardiac progenitor cells that can lead to CHD has not been systematically investigated at single cell resolution in vivo. Here, we integrated single-cell chromatin accessibility and transcriptomics to identify spatially restricted and temporally dynamic cREs in mouse heart development. We then defined dysregulated cREs in mice that lack Tbx1, a TF associated with CHD presentation in DiGeorge Syndrome. Integrated single-cell multiomics pointed to a critical role for Tbx1 in regulating cell-cell signaling pathways in second heart field progenitor cells but, surprisingly, not in differentiated OFT myocardium. Instead, we identified a specific subpopulation of neural crest cells that displayed aberrant expression of multiple members of Dlx, Fox and Etv transcription factors, which are required for patterning of outflow tract and craniofacial structures. Our work illustrates the power of single cell multiomics to uncover novel mechanistic insight into cardiac progenitor cells in CHD and paves the way for future in vitro perturbation studies targeting thousands of regulatory elements that may be involved in cardiogenesis.

Programmed Tube Bending Morphogenesis of a 3D Bioprinted Heart Tube

Jacqueline Bliley, Graduate Student at Carnegie Mellon University

Abstract: The cardiac tissue engineering field has emerged with the hope of generating whole heart organs. Contemporary approaches have focused on building the adult heart macroscopic organ structure using advanced biofabrication approaches; however, current engineered hearts display minimal contractility compared to the adult human heart. A limitation of these previous methods is the assumption that adult heart macroscopic structure will yield adult heart function. In contrast, in utero the heart develops from a linear tube that bends, loops and septates to form its three-dimensional structure and it is thought that these complex shape changes impart mechanical stresses that are critical to later heart organ structure and function. Thus, as an alternative approach, we sought to use embryonic heart morphogenesis as a guiding principle to develop whole heart organs. Here, we simulated the tube bending observed during early heart morphogenesis by inserting structural and mechanical asymmetries in 3D printed heart tubes to drive tube bending following application of cardiac fibroblast compaction forces. Heart tube bending resulted in region-specific changes in heart tube structure and function with the outer curvature of the bent tube displaying increased cardiomyocyte alignment and conduction velocity compared to the inner curvature of the bent tube. Differences in conduction paths were also observed with bent tubes initiating action potentials at the outer curvature, whereas linear tubes displayed conduction from one tube end to the other. These findings display some similarities to early heart morphogenesis and suggest that scientists should consider these morphogenetic stresses when attempting to build whole heart organs.

Umbilical vessels as potential grafts for congenital heart surgeries

Sae-Il Murtada, PhD, Associate Research Scientist at Yale University

Abstract: Infants born with a single ventricle defect undergo a series of staged surgeries over years to reconstruct the circulatory system. This allows deoxygenated blood from the veins to flow directly to the lungs without direct help from the heart. The earliest of these possible surgeries creates a shunt (called a Blalock-Taussig or BT-shunt) to route blood (from the subclavian artery) to the lungs (via a pulmonary artery) necessary for oxygenation. In order to design effective vascular grafts for use as BT-shunts, it is important to understand how mechanical properties of the graft affect vascular function over time. Here we characterized the mechanical properties of umbilical veins and arteries in mice and compared them to the subclavian and pulmonary arteries at two critical time points after birth. By using naturally occurring vessels rather than synthetic grafts, we reduce the risk of clotting and can also investigate the possibility of utilizing tissue-engineered grafts in the future. We found significant differences in mechanical properties in umbilical vessels and also regional differences between the subclavian and pulmonary arteries. Moreover, the umbilical veins and arteries displayed different contractile properties, which may have important implications when using umbilical vessels as potential grafts. We believe that these data can help improve the use of umbilical or tissue-engineered vessels as shunts in congenital heart surgeries and also lead to better mechanistic insights for human grafts in general.

Generating Artery and Vein Endothelial Cells From Pluripotent Stem Cells: A Toolkit for Stem Cell Biology and Tissue Engineering

Kevin Liu, Graduate Student at Stanford University

Abstract: The ability to generate human artery and vein endothelial cells (ECs) in vitro from human pluripotent stem cells (hPSCs) would provide a powerful platform to understand their diverse roles in health and disease and to engineer vascularized tissues. However, past efforts to convert hPSCs into ECs were lengthy (e.g., ~6-12 days of differentiation), inefficient (~10-60% of cells generated being endothelial cells), and typically generated cells that lacked clear artery or vein identity. Here we devise a strategy to generate human artery and vein ECs with high speed (within 3-4 days of hPSC differentiation) and purity (88-92% purity). Single-cell RNA-sequencing revealed stark transcriptional differences between hPSC-derived artery and vein ECs, confirming their distinct arteriovenous identities. Moreover hPSC-derived artery and vein ECs in vitro were transcriptionally similar to human fetal artery and vein ECs in vivo. Functionally, hPSC-derived artery and vein ECs could both form 3-dimensional networks in vivo and in vitro, but they differed in their functional responses to fluid flow (shear stress) and inflammation. There are thus extensive molecular and functional differences between hPSC-derived artery and vein ECs. To demonstrate the utility of these artery and vein ECs to model vascular diseases, we showed they could be infected by Biosafety-Level-4 (BSL4) viruses in vitro, which revealed new aspects of the tropism and effects of these fatal viruses.  In sum, the ability to generate artery and vein ECs with unprecedented speed and efficiency will advance regenerative medicine, tissue engineering, and the modeling of vascular diseases.

Perfusion Bioreactor System for Electromechanical Stimulation of Cardiac Tissue

Jessica Herrmann, Medical Student at Stanford University

Abstract: Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) offer a tantalizing future of patient-specific cardiac tissue for treating congenital heart defects. Achieving the highest possible level of function of iPSC-CMs is important for deriving clinically meaningful tissues. In particular, electromechanical stimulation has been shown to enhance the contractility and maturation of iPSC-CMs in small linear microtissues. Yet, to date, no bioreactor system has been designed for electromechanically maturing larger scale and perfusable cardiac tissues. Here, we report on the design, fabrication, and testing of a perfusion bioreactor system capable of applying combined mechanical and electrical stimulation to tubular iPSC-derived cardiac tissue constructs. The bioreactor is a closed-loop system consisting of the following components: the cellular media bath, which accommodates four tissue constructs with respective graphite electrodes; five cellular media reservoirs; external tubing to connect the media reservoirs to the media bath; a pump to promote flow through the tubing; a pressure box for mechanical stimulation; and circuitry for applying the mechanical and electrical stimulation regimes. Each cylindrical construct has its own independently addressable inlet for individual luminal perfusion, while a separate network of bifurcating channels permits the external perfusion of all four conduit outer walls simultaneously under identical flow conditions. Within the bioreactor system, cardiac tissue conduits containing fibroblasts, endothelial cells, and iPSC-derived cardiomyocytes were 3D bioprinted using freeform reversible embedding of suspended hydrogels (FRESH) and cultured over a period of 24 hours. Future work will involve immunohistochemical studies and RNA sequencing to examine the effects of varying electromechanical stimulation paradigms on tissue maturation.

Understanding Mechanisms of Atrial-Ventricular Specification and Differentiation During Early Cardiac Development Through Single Cell RNA Sequencing

David Gonzalez, MD/PhD Student at Icahn School of Medicine at Mount Sinai

Abstract: The molecular mechanisms driving atrial and ventricular fate acquisition in vivo are incompletely understood. We previously identified that transient expression of Foxa2 during gastrulation specifies cardiac progenitors that give rise to ventricular but not atrial myocytes. In order to understand transcriptional mechanisms underlying early atrial and ventricular specification prior to and during the morphogenetic events leading to chamber formation, we performed single-cell sequencing (scSeq) on sub-dissected cardiac regions from Foxa2-Cre;mTmG embryos at the cardiac crescent (E8.25), primitive heart tube (E8.75) and late heart tube (E9.25) stages. We found that Foxa2 lineage-traced cells can be identified transcriptionally by expression of EGFP without the need for cell sorting, allowing for comparison of atrial/ventricular specific progenitors at early stages. Through use of RNA velocity and lineage trajectory tools we found progression towards differentiated myocardial cell types occurs primarily based on heart field progenitor identity, and that different progenitor populations contribute to ventricular or atrial identity through separate differentiation mechanisms. We further show that in utero exposure to exogenous retinoic acid, which plays a role in atrial chamber specification and acts as a teratogen during development, causes defects in ventricular chamber size. scSeq of RA-exposed embryos demonstrated dysregulation in FGF signaling in anterior second heart field cells and a shunt in differentiation towards formation of head mesenchyme, as well as defects in cell-cycle exit in myocardial committed progenitors. In summary, combining our Foxa2 lineage traced model with scSeq of healthy and RA-injected embryos provides insight into transcriptional mechanisms underlying key events during atrial/ventricular differentiation.

10/06/21

Next Generation in Cardiac TE & SC: Early Career Spotlight

Getting to the Heart of the Matter: Complex Genetics and Congenital Heart Disease

Casey Gifford, PhD, Assistant Professor of Pediatrics (Cardiology) at Stanford University

Abstract: The heritable component of most common diseases is thought to be multigenic in nature, arising from the complex relationships of multiple variants that are not sufficient to cause disease in isolation but contribute to pathogenesis cooperatively. Recent evidence from our lab as well as others has suggested that multigenic mechanisms may underlie congenital heart disease. Here, I’ll discuss the efforts of my lab to use human induced pluripotent stem cell differentiation to dissect the complex genetic interactions that underlie heart development and likely contribute to congenital heart disease.

Advancing the Utility and Application of Engineered Human Cardiac Tissues

Sharon Fleischer, PhD, Postdoctoral Research Scientist at Columbia University, Laboratory for Stem Cells and Tissue Engineering (Gordana Vunjak-Novakovic Laboratory)

Abstract: Engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs) are increasingly used for drug discovery, pharmacological studies, and in modeling development and disease. Here, I will discuss the milliPillar platform, a robust and versatile technology that has been developed and validated to provide a streamlined pipeline for reproduction and utilization of engineered cardiac tissues for in vitro research. In addition, I will demonstrate the utility of the platform to diagnose myocarditis and improve heart disease risk stratification for precision medicine.

Bioprinting of Perfusable Cardiac Tissues at Therapeutic Scale

Sebastien Uzel, PhD, Research Associate at Harvard University (Jennifer Lewis Laboratory)

Abstract: The ability to biomanufacture vascularized human cardiac tissues, and ultimately, full heart ventricles for repair, replacement or regeneration from patient-specific cells is a grand challenge. My talk will highlight the various methods of bioprinting, biomaterials design, and tissue assembly that our team has developed to recreate the scale, cellular density, and function of cardiac tissues from human pluripotent stem cells.

09/22/21

Pioneers in Cardiac Tissue Engineering & Stem Cells

Creating a Pulmonary Valve that Grows with the Child: a Story of Discovery

Robert Tranquillo, PhD, Professor of Biomedical Engineering and Chemical Engineering & Materials Science at the University of Minnesota

Abstract: We have developed a biologically-engineered tube of cell-produced collagenous matrix, which is allogeneic upon a decellularization performed prior to implantation and thus “off-the-shelf.”  It is grown from donor dermal fibroblasts entrapped in a sacrificial fibrin hydrogel tube that is then decellularized using sequential detergent treatments. The resulting cell-produced matrix tube possesses physiological strength, compliance, alignment (circumferential) and growth potential, demonstrated in a growing lamb pulmonary artery replacement model because the matrix becomes a living tissue with the recipient’s cells post-implantation (Nat Comms 2016). Using the concept of a tubular heart valve, where the tube collapses inward with back-pressure between 3 equi-spaced constraints placed around the periphery to create one-way valve action, we have created a set of novel heart valves for adults and children that offer indefinite durability and growth potential, demonstrated by implantation in the growing lamb pulmonary artery for 52 weeks (Science 2021).

Evolution of Gene Regulatory Networks During Human Cardiogenesis

Eugin Destici, PhD, Assistant Project Scientist at the University of California San Diego (Neil Chi Laboratory)

Abstract: The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals.  Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined.  Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis.  These cis-regulatory elements are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts.  Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained cis-regulatory elements.  During early stages of human cardiogenesis, these cis-regulatory elements are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development.  In particular, we discovered that gained enhancers in the locus of the early developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role.  Genetic deletion of these enhancers resulted in not only reduced early cardiac gene expression but also decreased cardiomyocyte differentiation.  Overall, our results illuminate how human gained cis-regulatory elements may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain developmental roles through the evolutionary acquisition of enhancers.

09/08/21

State of the Science: Overview of Cardiac Tissue Engineering and Stem Cell Landscape

Stem Cells & Genomics for Precision Medicine

Joseph Wu, MD, PhD, Director of Stanford Cardiovascular Institute and Simon H. Stertzer, MD, Professor of Medicine and Radiology at Stanford University

Abstract: Recent technological advancements in multi-omics, CRISPR genome editing, and human induced pluripotent stem cells have enabled the implementation of precision medicine on an individual patient level. Here I will discuss recent advances in these technologies and how they may be used for elucidating mechanisms of cardiovascular diseases, for understanding chemotherapy-induced cardiotoxicity, and for implementing “clinical trial in a dish” concept.

Tissue Engineered Heart Valves: Where We’ve Been and Where We’re Going

John Mayer, Jr., MD, Senior Associate in Cardiac Surgery at Boston Children’s Hospital and Professor of Surgery at Harvard Medical School

Abstract: Investigations directed toward the development of a “tissue-engineered” heart valve began over 20 years ago. The initial concept was to create tissue engineered valve constructs based on biodegradable scaffolds combined with autologous cells, with the hopeful expectation that these living structures would prove to be durable and capable of growth. Early results in animals were promising, but scaling up for clinical application proved difficult. The field has evolved in several directions, and there are now initial clinical trials of acellular scaffolds as vascular conduits with the expectation that the host will repopulate these scaffolds. The history and current status of the field will be the topic of discussion.

03/03/21

Biology of Outcomes: Outcome Origins & Substrate-Outcome Relationship

Modeling ventricular hypoplasia in pulmonary atresia with intact ventricular septum (PA-IVS) using patient-specific induced pluripotent stem cells (iPSCs)

Mingtao Zhao, DVM, PhD and Vidu Garg, MD, Nationwide Children’s Hospital

Award Type: Innovation Fund

Abstract: Pulmonary atresia (PA) is a rare congenital heart defect (CHD) where the pulmonary valve that controls blood flow from the heart to the lungs does not properly develop. It is considered a critical CHD because surgical or catheter-based intervention is required soon after birth. In pulmonary atresia with intact ventricular septum (PA-IVS), the right ventricle (RV) does not fully develop because very little blood flows into or out of RV. PA-IVS is characterized by various degrees of RV hypoplasia and PA-IVS patients have long-term outcomes of single ventricle palliation (1v), 1 ½ ventricle palliation (1.5v), or bi-ventricular repair (2v). Mechanisms for the spectrum of RV hypoplasia in PA-IVS are unknown and difficult to fully ascribe to the absence of pulmonary valve. Currently, there are no reliable animal models to study the disease mechanisms of PA-IVS, further hindering the discovery of novel therapeutic treatments. In this study, we aim to elucidate the developmental mechanisms by which reduced biomechanical stretch suppresses cardiomyocyte proliferation and contributes to the RV hypoplasia found in PA-IVS patients. Our hypothesis is that reduced ventricular filling due to increased myocardial stiffness prevents embryonic cardiomyocyte proliferation and leads to the under development of RVs in PA-IVS patients. As human iPSC-derived cardiomyocytes (iPSC-CMs) are immature and resemble fetal-stage cardiomyocytes, we will employ PA-IVS patient-specific iPSC-CMs and engineered cyclic stretch to study how biomechanical forces alter the myocardial stiffness which further impacts early cardiomyocyte proliferation in PA-IVS.

The Genetics of Causes and Outcomes in SV CHD

Dr. Kim McBride, MD, Nationwide Children’s Hospital

Award Type: Innovation Fund

Abstract: The cause of  most SV CHD is not known, nor do we understand what influences the outcome. When there is an underlying genetic diagnosis, it is frequently made after many months, sometimes when complications or additional problems have surfaced. Making a diagnosis early can affect outcome and may give insight into the prognosis.  We will present how deep phenotyping and genome sequencing shortly after birth may impact care by early diagnosis.

 

02/17/21

Functional Cures: Bionic, Regenerative, & Transplantation Approaches

Piggy-back heart – a self powered Fontan assist device

Murali Padala, PhD, Emory University

Award Type: Innovation Fund

Abstract: Children surviving with a Fontan circulation lack a pump in their venous circulation, with the venous flow passively flowing into the lungs. A slightly higher pulmonary vascular resistance can impede venous flow, leading to its pooling in the vena cavae. Central venous pressure rises, and that has an adverse impact on the liver and several other organ systems. The need for mechanical devices that can assist the venous circulation in these patients is now established, but appropriate technologies are lacking that can be used in the long-term, without the risk of thrombosis and drive line infections, and do not make the child bed ridden. In this talk, I will introduce our concept of a piggy back heart, which is a self powered implantable chamber to drive Fontan circulation.

Development of a tissue engineered trans-catheter heart valve for use in the endovascular repair of single ventricle cardiac anomalies in utero

Dr Lakshmi Prasad Dasi, Georgia Tech

Award Type: Innovation Fund

Abstract: TBD

Unlocking Our Regenerative Capacity: Elucidating The Role Of LYST On Neotissue Formation In Tissue Engineered Vascular Grafts

Gabriel Mirhaidari, BS, The Abigail Wexner Research Institute at Nationwide Children’s Hospital / The Ohio State University

Award Type: Innovation Fund (Chris Breuer)

Abstract:  Tissue engineered vascular grafts (TEVGs) hold great promise for advancing the care of children born with single ventricle defects by providing an autologous vessel that can grow and remodel with the patient. However, TEVG stenosis remains a challenge to clinical translation. Our bench-to-beside and back again approach has focused on utilizing small animal models to better inform the biological mechanisms behind TEVG stenosis and identify potential therapeutic interventions for use in the clinic. We serendipitously discovered that TEVGs have improved performance when implanted in Beige mice, which are defined by a mutant gene encoding lysosomal trafficking regulator (LYST) protein, and were able to reproduce this effect through anti-LYST antibody treatment. As little is known about LYST and its immunomodulation effects, we herein report a series of experiments to further elucidate the role of LYST mediated immunomodulation in TEVG neovessel development. Wild-type (C57BL6/J) mice and Beige mice are implanted with TEVGs. At 2 weeks, mice are imaged utilizing micro-PET CT to determine degree of TEVG stenosis and inflammation. TEVGs are explanted and digested for single cell RNA-sequencing with follow-up hierarchical clustering, heat map and gene ontology and pathway analyses performed. An anti-LYST locked nucleic acid (LNA) siRNA is generated to allow selective silencing of LYST and subsequently its molecular signaling pathways associated with  LYST-mediated neovessel formation. An optimized dose of the LNA is administered to mice implanted with TEVGs with follow-up micro-PET CT angiography at 2-weeks to assess TEVG stenosis and inflammation. Grafts are explanted for single-cell RNA sequencing. The incidence of stenosis and inflammation, as well as results from single-cell RNA sequencing pathway analysis, is compared to results from the non LNA treated wild-type and beige mice to inform differences in molecular signaling pathways associated with LYST-mediated neotisue formation and LYST’s role in inhibiting TEVG stenosis.

02/03/21

Single Ventricle Clinical Sequelae: End-Organ Trajectory, Biomarkers, & Personalized Medicine

Origins and Burden of Multi-Organ Fibrosis in Patients with Single Ventricle Heart Disease

Elizabeth Goldmuntz, MD, FAAP, FACC and Jack Rychik, MD, Children’s Hospital of Philadelphia

Award Type: Innovation Fund

Abstract: Given medical and surgical advances, the majority of newborns presenting with single ventricle physiology now survive to early adulthood after staged surgical palliation to the Fontan circulation (FC).  However, time has shown that the ever-growing population with single ventricle disease (SVD) develops significant complications that impact morbidity and contribute to early mortality. Excessive cardiac and multi-organ fibrosis as a systemic process is likely an important contributor. The SVD population is exposed to multiple stimuli known to promote fibrogenesis including chronic hypoxia, and in the heart, pressure and volume loads. Additional mechanisms that promote fibrogenesis in other disease states, such as dysregulation of the renin-angiotensin-aldosterone (RAA) system and serotonin receptor signaling, may also contribute in the SVD population. Given notable variability in clinical outcomes, genetic variability that modifies individual response to clinical stressors likely plays an important role. Unfortunately, the underlying mechanisms of dysregulated fibrogenesis are poorly understood and we are unable to identify those at highest risk based on genetic or biochemical profiles.  Our goal is to identify genetic factors and biological mechanisms underlying this deleterious process so that the at-risk population can be identified and targeted therapeutics provided to prevent or reverse dysregulated fibrogenesis and preserve organ function. Specifically, we hypothesize that: (1) common and rare genetic variants within pathways regulating fibrogenesis contribute to individual risk, and (2) genotype and biomarker profiles will identify the SVD population at risk for mechanisms of dysregulated fibrogenesis, allowing for personalized, targeted therapeutics. To begin to identify the genetic and biological mechanisms underlying organ fibrogenesis, we will: (1) leverage a previously ascertained SVD cohort to test the association of genetic variants with myocardial fibrotic load as determined by cardiac MRI (CMR), and (2) prospectively enroll patients with SVD to better characterize multiorgan (heart, liver, kidney) fibrosis by MRI and serum biomarkers.

Shunt Audio Characteristics in Single Ventricle Infants as a Noninvasive Measure of Pulmonary Bloodflow

Saidie Rodriguez MD (other contributors: Camille Johnson, Goktug Ozmen, Omer Inan, PhD), Emory SOM and GaTech

Award Type: Innovation Fund

Abstract: Single ventricle infants with inadequate pulmonary blood flow require a systemic to pulmonary artery connection, (shunt) as initial palliation. When a patient is hypoxemic, shunt obstruction is among the differential diagnosis entertained with a high incidence of unplanned intervention. Current modalities to evaluate shunt function have limitations. There is a compelling need to innovate a noninvasive diagnostic tool to quantify the auditory characteristics of blood flow through shunts and could be done using a digital stethoscope combined with machine learning algorithms. We aim to 1) Establish audio characteristics of shunts correlating to a well-balanced circulation using various clinical parameters, and 2) Combine an acoustic signature with machine learning algorithms to improve detection of inadequate pulmonary bloodflow through the shunt.

01/06/21

Single Ventricle Etiology: Phenotype-Genotype Relationship & Model System Development

Ets1 in hypoplastic left heart syndrome

Shuyi Nie, Ph.D, Georgia Institute of Technology

Award Type: Innovation Fund

Abstract: Hypoplastic left heart syndrome (HLHS) is the most common cause of death in infants with congenital heart defects. Although it is clearly a genetic disease, little is known about the genetic mechanisms and pathophysiology underlying HLHS. In human, Ets1 has been identified as the genetic cause for heart defects in Jacobson Syndrome, in which HLHS is highly overrepresented. However, in mouse, loss of Ets1 leads to a series of heart defects including ventricular septal defects, non-compaction of left ventricle, and double-outlet right ventricle depending on the genetic backgrounds, but rarely HLHS. Recently, we showed that in frog, Ets1 loss leads to a heart phenotype very similar to that of human HLHS heart. The ventricular wall is thicker with a loss of ventricular trabeculation and a greatly reduced chamber size. There is also a subset of embryos with a growth arrested ventricle, suggesting that the development of HLHS-like phenotype may be a multi-step process. Using this model, we further demonstrated that Ets1 is particularly required in the cardiac mesoderm, rather than the cardiac neural crest cells for the heart development, consistent with an aortic arch artery contribution of the frog cardiac neural crest cells. Our ongoing work also indicates that the development of endocardium is impaired at Ets1 knocked down, suggesting that HLHS may result from a disrupted endocardium to myocardium signaling.

Modeling human TBX5 haploinsufficiency predicts regulatory networks for congenital heart disease:

Dr Irfan Kathiriya MD, PhD, University of California, San Francisco/Gladstone Institutes

Award Type:  Innovation Fund (Benoit Bruneau)

Abstract: Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD). However, underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways—including lineage decisions and genes associated with heart development, cardiomyocyte function, and CHD genetics—in discrete subpopulations of cardiomyocytes. Spatial transcriptomic mapping revealed chamber-restricted expression for many TBX5- sensitive transcripts. GRN analysis indicated that cardiac network stability, including vulnerable CHD-linked nodes, is sensitive to TBX5 dosage. A GRN-predicted genetic interaction betweenTbx5 and Mef2c was validated in mouse, manifesting as ventricular septation defects. These results demonstrate exquisite and diverse sensitivity to TBX5 dosage in heterogeneous subsets of iPSC-derived cardiomyocytes, and predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

11/11/20

Interventional Approaches

Comparative Hemodynamics of T-Junction and Y-Graft Anastomosis in the Penn State Fontan Pump

Nicolas Tobin

Pennsylvania State University, Department of Biomedical Engineering; Postdoc with Keefe Manning, PhD

Abstract: To improve outcomes in failing Fontan patients, Penn State is developing a small implanted centrifugal pump to support the heart demands and reduce pulmonary hypertension. The method of grafting the pump outlet onto the pulmonary arteries remains an important surgical consideration. Previous research has shown a Y-graft-type total cavopulmonary connection to have superior hemodynamics over a T-junction. This talk will discuss the comparative hemodynamics of a T-junction and Y-graft anastomosis of the Fontan pump outlet to patient-specific pulmonary arteries. Relevant hemodynamic parameters including dissipation of kinetic energy, wall shear stress, and pulmonary flow split will be presented.

Understanding Prenatal Brain Development in Single Ventricle to Improve Neurodevelopmental Outcome

Cynthia Ortinau, MD

Division of Newborn Medicine, Department of Pediatrics at Washington University in St. Louis School of Medicine; Assistant Professor of Pediatrics,

Abstract: Dramatic advances in the medical and surgical management of children born with single ventricle heart defects have enabled them to live longer and healthier lives. Yet, many survivors face substantial developmental delays or learning challenges that can have a major impact on long-term quality of life for children and their families. By adolescence, more than 2 out of every 3 children with single ventricle require developmental or special education services. Postnatal medical and surgical factors cannot predict which infants will face significant neurodevelopmental challenges. It is increasingly recognized that neurodevelopmental deficits result from abnormal brain development beginning during pregnancy. Our research in fetuses with congenital heart disease has shown smaller brain volumes and abnormal brain folding patterns occur by mid-gestation. Certain regions of the brain critical for long-term brain structure and function are particularly affected in fetuses with single ventricle heart disease. Abnormal prenatal brain development likely reflects several intersecting pathways. Factors such as reduced oxygen/nutrient delivery to the fetal brain from the heart defect itself, the function of the placenta, and the socioeconomic resources of the family may all impact prenatal brain development and neurodevelopment after birth. Our aim is to identify those clinical factors that improve mechanistic understanding of prenatal brain development in single ventricle and enable prediction of those at highest risk for neurodevelopmental impairments after birth. Our overarching goal is to use this information to facilitate prenatal counseling, provide targeted, early developmental services shortly after birth, and facilitate design and implementation of prenatal neuroprotective interventions.

 

10/28/20

Biomarker Discovery

Multi-Omic and Functional Metabolic Analysis Identified Dysregulated Lipid and Mitochondrial Metabolism in the Pediatric Failing Single Ventricle Heart

Anastacia (Tasha) Garcia, PhD

University of Colorado Anschutz Medical Campus, Children’s Hospital Colorado, Assistant Professor

Abstract: Congenital heart disease with single ventricle physiology (SV) encompasses a group of severe abnormalities in cardiac structure where improper development of the fetal heart results in only one functional pumping chamber. From a molecular standpoint, it is not well understood how the SV myocardium adapts to the chronic altered hemodynamic conditions of SV physiology, and cardiac dysfunction and ultimately heart failure (HF) are a common complication in the SV population. The purpose of this study was to characterize the transcriptomic, metabolomic, and lipidomic profiles in SV myocardium from both failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we conducted high-resolution respirometry (Oroboros Oxygraph system) to assess myocardial mitochondrial respiratory function in each of these populations. Lastly, we measured carnitine palmitoyltransferase (CPT) activity, a mitochondrial enzyme that allows the uptake of long-chain fatty acids for their subsequent oxidation. Multi-omics pathway analysis demonstrated multiple pathways that are similarly dysregulated in SVNF and SVHF, while pathways involved in mitochondrial and lipid metabolism were significantly dysregulated specifically in the SVHF population. Moreover, functional mitochondrial oxygen flux and CPT activity were significantly decreased in SVHF relative to BVNF controls. Therefore, these results provide new insights into SVHF by identifying unique gene, metabolite and lipid changes, including those related to mitochondrial metabolic function, which may serve as potential therapeutic targets for the treatment or prevention of HF in the SV population. These data are corroborated by the significant decrease seen in functional assessments of mitochondrial oxygen flux and CPT activity.  Based on these findings, we have also recently begun to explore circulating peripheral mononuclear cells a potential biomarkers of myocardial mitochondrial function in the SV population. We propose that peripheral blood mononuclear cells (PBMCs) isolated from children with SV could serve as surrogate circulating molecular biomarkers for myocardial mitochondrial alterations and provide predictive prognostic value to this unique patient population. Our preliminary data demonstrates PBMCs from SVHF patients display significant changes in mitochondrial oxygen flux manifesting in decreased respiratory capacity, ATP production, and coupling efficiency, and increased reactive oxygen species relative to controls. Therefore, we hypothesize that myocardial mitochondrial metabolism represents a biomarker of disease progression in SV patients, and that patient-derived PBMCs serve as a proxy for myocardial cellular respiration.

Predicting Outcomes in Single Ventricle Heart Disease Through Circulating miRNAs

Stephanie Nakano, MD

Assistant Professor, Pediatric Cardiology; Children’s Hospital Colorado

Abstract: Single ventricle congenital heart disease (SV) is universally fatal without intervention and is the leading cause of cardiovascular death in infancy. Currently, the most common management strategy for SV is a series of palliative surgeries. While these surgeries represent significant advances in medical care, mortality remains high, with 30% of patients dying in the first year of life. Importantly, the ability to predict which infants will do well with surgical palliation is lacking.

Circulating miRNAs are increasingly recognized as effective biomarkers in a broad range of medical disciplines, aiding in both diagnosis and prognosis. Our hypothesis was that circulating miRNA profiles prior to surgical palliation may correlate with SV survival at one year and assist with risk-stratification in this population. Serum samples from subjects with SV (of right ventricular morphology) were obtained at the following time points: Pre-Norwood (n=71), Pre-Glenn (n=46) and Pre-Fontan (n=25). Outcomes were classified as alive versus death or heart transplant listing by one year of age. Serum was subject to three freeze/heat cycles to maximize miRNA release, then miRNAs were reverse transcribed using a pool of primers specific for each miRNA. Real-time PCR was performed in 384-well plates containing sequence-specific primers and TaqMan probes in the ABI7900HT. Analysis of Pre-Norwood samples demonstrated downregulation of miR-15b, -192, and 193b in patients who died or required heart transplant listing (n=22) compared to those who were alive (n=49) at one year of age. Additionally, miR-let-7b, -26a, and -454 were differentially expressed between the Pre-Norwood, Pre-Glenn, and Pre-Fontan groups, suggesting an association with surgical stage and/or age. Circulating miRNA profiles are distinct in pediatric SV patients at each surgical stage, and demonstrate promise as prognostic biomarkers of 1-year outcome in the Pre-Norwood SV population.

10/14/20

Tissue Engineering Approaches & Applications

Engineering In Vitro Vascularized Tissues Using FRESH 3D Bioprinted Collagen Scaffolds

Andrew Hudson, MS

Carnegie Mellon University, Regenerative Bioaterials Therapeutic Group (Adam Feinberg PhD), PhD Student

Abstract: One of the foremost challenges remaining to engineer large (>1 cm3), transplantable tissues is creating the microvasculature necessary to promote cell viability and eventual tissue function. Given the intricate 3D architecture of native tissues, 3D bioprinting aims to build complex geometries that would otherwise be unobtainable through traditional methods, seeking to create viable tissues and organs. Two major hurdles to bioprinting large tissues are 3D printing extracellular matrix (ECM) proteins at high fidelity and generating the microvasculature necessary to maintain cell viability. The mechanical instability of soft (E < 150 kPa) ECM proteins causes significant deformation during printing that hinders the immediate creation of patent microvasculature. Recently, improvements to our 3D bioprinting method Freeform Reversible Embedding of Suspended Hydrogels (FRESH) have allowed us to print ECM components like collagen with a high degree of geometric complexity at a fidelity down to 20 μm.

With a major obstacle largely addressed, we have begun to work towards developing an in vitro vascularization system for vessel templates FRESH printed from collagen. This system is based on leveraging two phenomena in tissue engineering – microporosity and cellular remodeling. By increasing tissue scaffold porosity, we show an increase rate of diffusion through a collagen matrix translates to improved cellular viability while in vivo data shows porosity improves host-driven angiogenesis. By introducing endothelial cells to our porous bioprinted vessel templates, we seek to create an in vitro vascularization system that results in a large, vascularized tissue for transplantation.

Dynamic Loading of Human Engineered Heart Tissue Enhances Contractile Function and Drives Desmosome-linked Disease Phenotype

Jaci Billey, MS

Carnegie Mellon University, Regenerative Biomaterials Therapeutic Group (Adam Feinberg PhD), PhD Student

Abstract: Heart failure is a significant concern affecting more than 5 million adults in the U.S. alone. Hemodynamic loads, including preload (stretch on heart muscle) and afterload (pressure the heart must work against to eject blood), can lead to maladaptive structural and functional changes in the heart, specifically ventricular dilation and reduced force generation. Engineered heart tissues (EHTs) have the potential to provide insight into loading-induced disease progression but current 3D EHT approaches are constrained (length is held constant), which limits their ability to model the altered loads experienced during heart failure, the tissue morphological changes that occur, and the consequences that these have on heart muscle tissue force generation. Here, we have developed a dynamic EHT (dyn-EHT) model that addresses these limitations by integrating EHTs with an elastic polydimethylsiloxane (PDMS) strip, which provides active preload and a contractile force measurement based on strip bending during EHT contraction. Our results demonstrate that dynamic loading is beneficial in wild-type EHTs leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC–derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke a disease phenotype (tissue lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output.

09/30/20

Mathematical and Computation Models

Interpretable machine learning for biological discovery

Susan Liao, PhD

NYU Courant Institute of Mathematical Sciences, Postdoctoral Fellow with Jef Boeke, PhD, and Additional Ventures LSRF Fellow

Abstract: Neural networks hold great promise for deciphering complex biological logic due to their expressive nature that can describe a breadth of relationships in data that can be quantitatively described through mathematical functions. However, despite great success with predicting outcomes, most neural networks fail to advance biological discovery as they were designed with primary goal of prediction accuracy. Despite excellent predictions, the vast majority of neural networks cannot explain how they arrived these predictions. It is difficult to trust or verify whether these predictions reflect true biological relationships or instead capture biases in the dataset. Uninterpretable neural networks thus provide limited mechanistic insight into underlying biological processes. Furthermore, predictions from an uninterpretable network may not generalize, failing to predict outcomes from data that it has not seen before, limiting the utility of the network in basic research or clinical settings. There is a clear need to design interpretable neural networks that will enable biologists to generate and test novel hypotheses from their data. In this presentation I will present our progress towards this goal, focusing on how advances in data acquisition and data analysis can improve neural network interpretability. These innovations will address interpretability concerns across a wide range of biological questions involving gene expression in both development and disease.

Roles of negative and positive feedback in cardiovascular adaptations

Linda Irons, PhD

Department of Biomedical Engineering, Yale University, Postdoctoral fellow with Jay Humphrey, PhD

Abstract: Single ventricle defects result in altered hemodynamics and reduced blood oxygenation, both of which can stimulate remodeling responses throughout the systemic and pulmonary circulations. Such changes, in turn, can adversely affect the delivery of oxygenated blood within end organs at appropriate pressures and flows, hence contributing to progressive morbidity and mortality. There is, therefore, a pressing need to understand not just how the hemodynamics differs in single ventricle disease, but also how these alterations drive widespread remodeling that is often maladaptive. Toward this end, we have developed computational models “from transcript to tissue” to simulate how cell-perceived stimuli result in particular transcriptional changes that in turn drive tissue level changes in structure and function. Whereas homeostatic processes drive adaptive remodeling through negative feedback, compromised or lost hemostasis results in maladaptive responses characterized by positive feedback.

09/16/20

Stem Cell Models and Applications

Roadmap to efficiently generate human artery and vein endothelial cells from pluripotent stem cells

Kyle Loh, PhD

Stanford University, Assistant Professor and The Anthony DiGenova Endowed Faculty Scholar

Abstract: Endothelial cells—encompassing molecularly-distinct arteries versus veins—pervade all tissues and have manifold roles in health and disease, and also likely contribute to congenital heart defects. What instigates human endothelial cell diversity and how can we recreate such diversity in vitro? Here we efficiently differentiate human pluripotent stem cells through a sequence of branching lineage choices into artery or vein endothelial cells, within 3-4 days. This roadmap encompasses the stepwise changes in extracellular signals, gene expression, chromatin state and cell-surface markers during specialization of artery vs. vein cells from their mesodermal precursors, including a two-step process for vein specification. This thus provides an informative reference map for human artery and vein development. The newfound capability to rapidly and efficiently generate human artery-specific and vein-specific endothelial cells en masse should avail tissue engineering, disease modeling and other diverse applications that hinge on a large-scale supply of human endothelial cells.

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients

Serah Kang, PhD

UCSF & Gladstone Institutes, Postdoctoral Fellow (McDevitt Lab)

Abstract: Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19.

09/02/20

Genetics and Regulatory Elements

Predicting the disruption of 3D genome folding in congenital heart defects

Maureen Pittman, BS, UCSF & Gladstone Institutes, PhD Student with Katie Pollard, PhD

Abstract: Many disease-associated genetic variants are located in putative regulatory elements, suggesting a major role for transcriptional misregulation in disease. This is especially relevant to developmental disorders, in which the disruption of finely-tuned transcriptional networks is likely to have acute consequences to cell differentiation and morphogenesis. The fidelity of gene regulation is enforced partly by the three-dimensional organization of the genome, which is arranged such that regulatory elements and their target promoters are able to make contact in the appropriate contexts. Structural variants have the potential to disrupt that system by changing the contact frequency of promoters to their regulatory elements. Here, we hypothesize that such variants may contribute to the formation of congenital heart defects. Using structural variants identified by the Pediatric Cardiac Genomics Consortium, we use a convolutional neural network to predict the resulting change in chromatin contact frequency and prioritize variants for experimental validation.

Genome-wide maps of enhancer regulation connect risk variants to disease genes

Jesse Engreitz, PhD, Stanford University, Assistant Professor

Abstract: Enhancers harbor thousands of variants associated with common diseases and traits. Each of these variants could reveal insights into disease mechanisms or therapeutic targets. Yet, it has proven difficult to connect these variants to their molecular functions because we have lacked tools to systematically map which enhancers regulate which genes in which cell types. Here we use new CRISPR methods to perturb >4,500 enhancer-gene connections in several cell types. We show that an Activity-by-Contact (ABC) Model of enhancer function — involving multiplying enhancer activity by enhancer-promoter contact — can accurately predict these experimental perturbations based on easily obtained maps of chromatin state. We apply this ABC Model to create enhancer-gene maps in 131 cell types and tissues, and use these maps to interpret the functions of genetic variants associated with common diseases and complex traits. We find that over half of causal noncoding GWAS variants likely act via effects on enhancers. Variants associated with cardiovascular diseases connect to genes in endothelial cells and cardiomyocytes. These ABC maps provide a generalizable strategy to connect common disease risk variants in enhancers to target genes, and will help to understand the genetic etiology of congenital heart defects.

We're sorry, there are no results for your query. Please try again.

Show more
Show less