Highlights d Functional VSMCs could be efficiently generated on a large scale from hiPSCs d Optimized biochemical and biophysical conditions were used to generate hiPSC-TEVGs d hiPSC-TEVGs presented mechanical strength comparable to that of saphenous veins d hiPSC-TEVGs maintained patency and mechanical function following rat implantation

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.

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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.

Graphical abstractEnergy yielding pathways of human mesenchymal stem cellsGlucose is consumed by both the glycolytic and the oxidative pathways. Pyruvate, acetoacetate and glutamine are oxidised by hMSCs. Exogenous substrates are shown in boxes with solid lines, pathway intermediates in boxes with dashed lines and products in boxes with dotted lines. Arrows showing routes of entry of exogenous substrates into pathways are weighted to represent rates of utilisation (not to scale).

peroxisome proliferator activated receptor β/δ (PPARβ/δ) has pro-angiogenic functions, but whether PPARβ/δ modulates endothelial cell metabolism to support the dynamic phenotype remains to be established. This study characterised the metabolic response of HUVEC to the PPARβ/δ agonist, GW0742, and compared these effects with those induced by VEGF-A. In HUVEC monolayers, flux analysis revealed that VEGF-A promoted glycolysis at the expense of fatty acid oxidation (FAO), whereas GW0742 reduced both glycolysis and FAO. Only VEGF-A stimulated HUVEC migration and proliferation whereas both GW0742 and VEGF-A promoted tubulogenesis. Studies using inhibitors of PPARβ/δ or sirtuin-1 showed that the tubulogenic effect of GW0742, but not VEGF-A, was PPARβ/δand sirtuin-1-dependent. HUVEC were reliant on glycolysis and FAO, and inhibition of either pathway disrupted cell growth and proliferation. VEGF-A was a potent inducer of glycolysis in tubulogenic HUVEC, while FAO was maintained. In contrast, GW0742-induced tubulogenesis was associated with enhanced FAO and a modest increase in glycolysis. These novel data reveal a context-dependent regulation of endothelial metabolism by GW0742, where metabolic activity is reduced in monolayers but enhanced during tubulogenesis. These findings expand our understanding of PPARβ/δ in the endothelium and support the targeting of PPARβ/δ in regulating ec behaviour and boosting tissue maintenance and repair. Endothelial cells (ECs) lining normal vessels remain relatively quiescent, but retain the remarkable ability to acquire a highly active and dynamic phenotype following tissue injury or ischaemia 1. This is essential for angiogenesis, during which ECs sprout, migrate and proliferate to establish a new vascular network. Cells undergoing such rapid and dynamic alterations in growth and proliferation are known to exhibit significant alterations in their metabolism 2 , so interest in targeting master metabolic regulators, or metabolic pathways directly, to influence EC behaviour is growing 3,4. While the importance of various metabolic pathways for sustaining angiogenic behaviour is increasingly recognised 3,5 , knowledge of how ECs adapt their metabolism in response to extrinsic signals is particularly lacking. Exposure to vascular endothelial growth factor A (VEGF-A), a well-studied angiogenic growth factor, is known to increase glycolytic flux in ECs, in part through stimulation of the phosphoinositide 3-kinase (PI3-K)/ Akt signalling pathway and increased activity of key enzymes in the glycolytic cascade 6. Characterisation of the metabolic response of ECs to other established and putative angiogenic factors, and the functional importance of these changes, has not been addressed to date. The peroxisome proliferator activated receptor β/δ (PPARβ/δ) nuclear receptor has been identified as a facilitator of the angiogenic response and synthetic PPARβ/δ agonists generally exhibit pro-angiogenic activity 7-10. PPARβ/δ controls the expression of multiple genes linked to the regulation of glucose ...

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)

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