Pushing the envelope in tissue engineering: Ex vivo production of thick vascularized cardiac extracellular matrix constructs

Functional vascularization is a prerequisite for cardiac tissue engineering of constructs with physiological thicknesses. We previously reported the successful preservation of main vascular conduits in isolated thick acellular porcine cardiac ventricular ECM (pcECM). We now unveil this scaffold's potential in supporting human cardiomyocytes and promoting new blood vessel development ex vivo, providing long-term cell support in the construct bulk. A custom-designed perfusion bioreactor was developed to remodel such vascularization ex vivo, demonstrating, for the first time, functional angiogenesis in vitro with various stages of vessel maturation supporting up to 1.7 mm thick constructs. A robust methodology was developed to assess the pcECM maximal cell capacity, which resembled the human heart cell density. Taken together these results demonstrate feasibility of producing physiological-like constructs such as the thick pcECM suggested here as a prospective treatment for end-stage heart failure. Methodologies reported herein may also benefit other tissues, offering a valuable in vitro setting for "thick-tissue" engineering strategies toward large animal in vivo studies.Israeli Science Foundation/1563/10Singapore National Research Foundatio

Engineering 3D thick porcine extracellular matrix for cardiac regeneration

This project focused on engineering a thick cardiac patch using an acellular scaffold derived from a porcine left ventricle as a possible replacement therapy for the scar tissue formed following acute heart damage. Whereas most studies demonstrated cardiac constructs with thicknesses up to a few hundred micrometers, this project aims to recellularize an acellular scaffold with comparable thickness to the human native heart (1-1.2 cm).DOCTOR OF PHILOSOPHY (MSE

A mathematical model predicting the coculture dynamics of endothelial and mesenchymal stem cells for tissue regeneration

In most tissue engineering applications, understanding the factors affecting the growth dynamics of coculture systems is crucial for directing the population toward a desirable regenerative process. Yet, no comprehensive analysis method exists to quantify coculture population dynamics, let alone, a unifying model addressing the “environmental” factors influencing cell growth, all together. Here we suggest a modification of the Lotka-Volterra model to analyze the population dynamics of cocultured cells and predict their growth profiles for tissue engineering applications. This model, commonly used to describe the population dynamics of a prey and predator sharing a closed ecological niche, was found to fit our empirical data on cocultures of endothelial cells (ECs) and mesenchymal stem cells (MSCs) that have been widely investigated for their regenerative potential. Applying this model to cocultures of this sort allows us to quantify the effect that culturing conditions have on the way cell growth is affected by the same cells or by the other cells in the coculture. We found that in most cases, EC growth was inhibited by the same cells but promoted by MSCs. The principles resulting from this analysis can be used in various applications to guide the population toward a desired direction while shedding new light on the fundamental interactions between ECs and MSCs. Similar results were also demonstrated on complex substrates made from decellularized porcine cardiac extracellular matrix, where growth occurred only after coculturing ECs and MSCs together. Finally, this unique implementation of the Lotka-Volterra model may also be regarded as a roadmap for using such models with other potentially regenerative cocultures in various applications.Published Versio

Novel Mechanism for an Old Drug: Phenazopyridine is a Kinase Inhibitor Affecting Autophagy and Cellular Differentiation

Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the two phosphatidylinositol kinases PI4KB and PIP4K2C, the latter being known for participating in pain induction. Docking revealed that phenazopyridine forms strong H-bonds with the hinge region of the ATP-binding pocket of these kinases. As previous studies suggested increased autophagy upon inhibition of the phosphatidyl-inositol/AKT pathway, we also investigated the impact of phenazopyridine on this pathway and found an upregulation. In conclusion, our study demonstrates for the first time that phenazopyridine is a kinase inhibitor, impacting notably phosphatidylinositol kinases involved in nociception