Tasneem Ebrahim’s research while affiliated with Icahn School of Medicine at Mount Sinai and other places

Purpose of the Review This review aims to discuss the process of cardiomyocyte maturation, with a focus on the underlying molecular mechanisms required to form a fully functional heart. We examine both long-standing concepts associated with cardiac maturation and recent developments, and the overall complexity of molecularly integrating all the processes that lead to a mature heart. Recent Findings Cardiac maturation, defined here as the sequential changes that occurring before the heart reaches full maturity, has been a subject of investigation for decades. Recently, there has been a renewed, highly focused interest in this process, driven by clinically motivated research areas where enhancing maturation may lead to improved therapeutic opportunities. These include using pluripotent stem cell models for cell therapy and disease modeling, as well as recent advancements in adult cardiac regeneration approaches. Summary We highlight key processes underlying maturation of the heart, including cellular and organ growth, and electrophysiological, metabolic, and contractile maturation. We further discuss how these processes integrate and interact to contribute to the overall complexity of the developing heart. Finally, we emphasize the transformative potential for translating relevant maturation concepts to emerging models of heart disease and regeneration.

The specification of distinct cardiac lineages occurs prior to chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here we performed single cell analysis at the cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics utilizing computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

Migration is an important function for natural killer cells. Cell motility has implications in development, tissue infiltration, and cytotoxicity, and measuring the properties of natural killer (NK) cell migration using in vitro assays can be highly informative. Many researchers have an interest in studying properties of NK cell migration in the context of genetic mutation, disease, or in specific tissues and microenvironments. Motility assays can also provide information on the localization of proteins during different phases of cell migration. These assays can be performed on different surfaces for migration or coupled with chemoattractants and/or target cells to test functional outcomes or characterize cell migration speeds and phenotypes. NK cells undergo migration during differentiation in tissue, and these conditions can be modeled by culturing NK cells on a confluent bed of stromal cells on glass and imaging cell migration. Alternatively, fibronectin- or ICAM-1-coated surfaces promote NK cell migration and can be used as substrates. Here, we will describe techniques for the experimental setup and analysis of NK cell motility assays by confocal microscopy or in-incubator imaging using commercially available systems. Finally, we describe open-source software for analyzing cell migration using manual tracking or automated approaches and discuss considerations for the implementation of each of these methods.

The specification and differentiation of atrial and ventricular myocardial cell types during development is incompletely understood. We have previously shown that Foxa2 expression during gastrulation identifies a population of ventricular fated progenitors, allowing for labeling of these cells prior to the morphogenetic events that lead to chamber formation and acquisition of bona fide atrial or ventricular identity. In this study, we performed single cell RNA sequencing of Foxa2Cre;mTmG embryos at the cardiac crescent (E8.25), primitive heart tube (E8.75) and heart tube (E9.25) stage in order to understand the transcriptional mechanisms underlying formation of atrial and ventricular cell types at the earliest stages of cardiac development. We find that 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 identified a number of candidate markers that define such differentiation processes, as well as differential regulation of metabolic processes that distinguish atrial and ventricular fated cells at the earliest stages of development. We further show that exogenous injection with retinoic acid during formation of the cardiac primordia causes defects in ventricular chamber size and is associated with dysregulation in FGF signaling in anterior second heart field cells and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit in myocardial committed progenitors that result in formation of hypomorphic ventricles with decreased expression of important metabolic processes and sarcomere assembly. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac progenitor cell specification and differentiation, and the precise effects of manipulating cardiac progenitor field patterning via exogenous retinoic acid signaling.