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.