De Novo Elastin Assembly Alleviates Development of Supravalvular Aortic Stenosis.

BACKGROUND: A series of incurable cardiovascular disorders arise due to improper formation of elastin during development. Supravalvular aortic stenosis (SVAS), resulting from a haploinsufficiency of ELN, is caused by improper stress sensing by medial vascular smooth muscle cells, leading to progressive luminal occlusion and heart failure. SVAS remains incurable, as current therapies do not address the root issue of defective elastin. METHODS: We use SVAS here as a model of vascular proliferative disease using both human induced pluripotent stem cell-derived vascular smooth muscle cells and developmental Eln± mouse models to establish de novo elastin assembly as a new therapeutic intervention. RESULTS: We demonstrate mitigation of vascular proliferative abnormalities following de novo extracellular elastin assembly through the addition of the polyphenol epigallocatechin gallate to SVAS human induced pluripotent stem cell-derived vascular smooth muscle cells and in utero to Eln± mice. CONCLUSIONS: We demonstrate de novo elastin deposition normalizes SVAS human induced pluripotent stem cell-derived vascular smooth muscle cell hyperproliferation and rescues hypertension and aortic mechanics in Eln± mice, providing critical preclinical findings for the future application of epigallocatechin gallate treatment in humans.

Epigallocatechin gallate facilitates extracellular elastin fiber formation in induced pluripotent stem cell derived vascular smooth muscle cells for tissue engineering.

Tissue engineered vascular grafts possess several advantages over synthetic or autologous grafts, including increased availability and reduced rates of infection and thrombosis. Engineered grafts constructed from human induced pluripotent stem cell derivatives further offer enhanced reproducibility in graft production. One notable obstacle to clinical application of these grafts is the lack of elastin in the vessel wall, which would serve to endow compliance in addition to mechanical strength. This study establishes the ability of the polyphenol compound epigallocatechin gallate, a principal component of green tea, to facilitate the extracellular formation of elastin fibers in vascular smooth muscle cells derived from human induced pluripotent stem cells. Further, this study describes the creation of a doxycycline-inducible elastin expression system to uncouple elastin production from vascular smooth muscle cell proliferative capacity to permit fiber formation in conditions conducive to robust tissue engineering.

Physiological and pharmacological stimulation for in vitro maturation of substrate metabolism in human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable human cardiac cells to be studied in vitro, although they use glucose as their primary metabolic substrate and do not recapitulate the properties of adult cardiomyocytes. Here, we have explored the interplay between maturation by stimulation of fatty acid oxidation and by culture in 3D. We have investigated substrate metabolism in hiPSC-CMs grown as a monolayer and in 3D, in porous collagen-derived scaffolds and in engineered heart tissue (EHT), by measuring rates of glycolysis and glucose and fatty acid oxidation (FAO), and changes in gene expression and mitochondrial oxygen consumption. FAO was stimulated by activation of peroxisome proliferator-activated receptor alpha (PPARα), using oleate and the agonist WY-14643, which induced an increase in FAO in monolayer hiPSC-CMs. hiPSC-CMs grown in 3D on collagen-derived scaffolds showed reduced glycolysis and increased FAO compared with monolayer cells. Activation of PPARα further increased FAO in cells on collagen/elastin scaffolds but not collagen or collagen/chondroitin-4-sulphate scaffolds. In EHT, FAO was significantly higher than in monolayer cells or those on static scaffolds and could be further increased by culture with oleate and WY-14643. In conclusion, a more mature metabolic phenotype can be induced by culture in 3D and FAO can be incremented by pharmacological stimulation.

Modular design of a tissue engineered pulsatile conduit using human induced pluripotent stem cell-derived cardiomyocytes.

Single ventricle heart defects (SVDs) are congenital disorders that result in a variety of complications, including increased ventricular mechanical strain and mixing of oxygenated and deoxygenated blood, leading to heart failure without surgical intervention. Corrective surgery for SVDs are traditionally handled by the Fontan procedure, requiring a vascular conduit for completion. Although effective, current conduits are limited by their inability to aid in pumping blood into the pulmonary circulation. In this report, we propose an innovative and versatile design strategy for a tissue engineered pulsatile conduit (TEPC) to aid circulation through the pulmonary system by producing contractile force. Several design strategies were tested for production of a functional TEPC. Ultimately, we found that porcine extracellular matrix (ECM)-based engineered heart tissue (EHT) composed of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and primary cardiac fibroblasts (HCF) wrapped around decellularized human umbilical artery (HUA) made an efficacious basal TEPC. Importantly, the TEPCs showed effective electrical and mechanical function. Initial pressure readings from our TEPC in vitro (0.68 mmHg) displayed efficient electrical conductivity enabling them to follow electrical pacing up to a 2 Hz frequency. This work represents a proof of principle study for our current TEPC design strategy. Refinement and optimization of this promising TEPC design will lay the groundwork for testing the construct's therapeutic potential in the future. Together this work represents a progressive step toward developing an improved treatment for SVD patients. STATEMENT OF SIGNIFICANCE: Single Ventricle Cardiac defects (SVD) are a form of congenital disorder with a morbid prognosis without surgical intervention. These patients are treated through the Fontan procedure which requires vascular conduits to complete. Fontan conduits have been traditionally made from stable or biodegradable materials with no pumping activity. Here, we propose a tissue engineered pulsatile conduit (TEPC) for use in Fontan circulation to alleviate excess strain in SVD patients. In contrast to previous strategies for making a pulsatile Fontan conduit, we employ a modular design strategy that allows for the optimization of each component individually to make a standalone tissue. This work sets the foundation for an in vitro, trainable human induced pluripotent stem cell based TEPC.

Metabolic flux analyses to assess the differentiation of adult cardiac progenitors after fatty acid supplementation.

Myocardial infarction is the most prevalent of cardiovascular diseases and pharmacological interventions do not lead to restoration of the lost cardiomyocytes. Despite extensive stem cell therapy studies, clinical trials using cardiac progenitor cells have shown moderate results. Furthermore, differentiation of endogenous progenitors to mature cardiomyocytes is rarely reported. A metabolic switch from glucose to fatty acid oxidation occurs during cardiac development and cardiomyocyte maturation, however in vitro differentiation protocols do not consider the lack of fatty acids in cell culture media. The aim of this study was to assess the effect of this metabolic switch on control and differentiated adult cardiac progenitors, by fatty acid supplementation. Addition of oleic acid stimulated the peroxisome proliferator-activated receptor alpha pathway and led to maturation of the cardiac progenitors, both before and after transforming growth factor-beta 1 differentiation. Addition of oleic acid following differentiation increased expression of myosin heavy chain 7 and connexin 43. Also, total glycolytic metabolism increased, as did mitochondrial membrane potential and glucose and fatty acid transporter expression. This work provides new insights into the importance of fatty acids, and of peroxisome proliferator-activated receptor alpha, in cardiac progenitor differentiation. Harnessing the oxidative metabolic switch induced maturation of differentiated endogenous stem cells. (200 words).

Changing Metabolism in Differentiating Cardiac Progenitor Cells-Can Stem Cells Become Metabolically Flexible Cardiomyocytes?

The heart is a metabolic omnivore and the adult heart selects the substrate best suited for each circumstance, with fatty acid oxidation preferred in order to fulfill the high energy demand of the contracting myocardium. The fetal heart exists in an hypoxic environment and obtains the bulk of its energy via glycolysis. After birth, the "fetal switch" to oxidative metabolism of glucose and fatty acids has been linked to the loss of the regenerative phenotype. Various stem cell types have been used in differentiation studies, but most are cultured in high glucose media. This does not change in the majority of cardiac differentiation protocols. Despite the fact that metabolic state affects marker expression and cellular function and activity, the substrate composition is currently being overlooked. In this review we discuss changes in cardiac metabolism during development, the various protocols used to differentiate progenitor cells to cardiomyocytes, what is known about stem cell metabolism and how consideration of metabolism can contribute toward maturation of stem cell-derived cardiomyocytes.

Strengthening STEM Education through Community Partnerships.

California State University San Marcos (CSUSM) and San Marcos Elementary Schools have established a partnership to offer a large-scale community service learning opportunity to enrich science curriculum for K-5 students. It provides an opportunity for science, technology, engineering, and math (STEM) majors to give back to the community, allowing them to experience teaching in an elementary classroom setting, in schools that lack the resources and science instructor specialization needed to instill consistent science curricula. CSUSM responded to this need for more STEM education by mobilizing its large STEM student body to design hands-on, interactive science lessons based on Next Generation Science Standards (NGSS). Since 2012, the program has reached out to over four thousand K-5 students, and assessment data have indicated an increase in STEM academic performance and interest.