Abstract
1641
Objectives Human induced pluripotent cells (hiPSC) have been shown to represent an unlimited source of spontaneously contracting cardiomyocytes (CM) by applying appropriate differentiation protocols. A bottleneck of induced cardiomyocyte technology for practical application is the difficulty in establishing standardized differentiation strategies for homogeneous and fully maturated cardiomyocytes. Although hiPSC-CM share various characteristic hallmarks with endogenous cardiomyocytes, it remains a question as to what extent metabolic characteristics are equivalent to mature mammalian cardiomyocytes. Here we set out to functionally characterize the metabolic status of hiPSC-CM in vitro employing a radionuclide tracer uptake assay.
Methods The hiPSCs were obtained by transgene-free technology. Cardiac differentiation of hiPSC was induced using a combination of well-orchestrated extrinsic stimuli such as BMP4 and WNT activation followed by WNT inhibition and lactate based cardiomyocyte enrichment. Differentiation was confirmed by immunostaining for α-actinin and cardiac Troponin T. For characterization of metabolic substrates, dual tracer uptake studies were performed with 18F-2-fluoro-2-deoxy-D-glucose (FDG) and 125I-β-methyl-iodophenyl-pentadecanoic acid (BMIPP) as transport markers of glucose and fatty acids, respectively.
Results Cardiac differentiation were confirmed by immunofluorescence staining both against α-actinin and cardiac Troponin T after maturation and enrichment process. Glucose (FDG) uptake of hiPSC-CM was significantly lower than that of undifferentiated hiPS cells while fatty acid (BMIPP) uptake was significantly increased in hiPSC-CM (BMIPP/ FDG tracer uptake ratio in hiPSC-CM and hiPSC = 8.58±1.67 and 0.90±0.06, respectively. P<0.0001). Immunostaining confirmed expression of fatty acid transport (SLC27) and fatty acid binding protein (FABP) after cardiac differentiation.
Conclusions After cardiac differentiation of human induced pluripotent stem cells, in vitro tracer uptake assays confirmed enhanced cellular intake of fatty acids which are known as a main and unique energy source of mammalian hearts in-vivo. Our study sheds a new light on the metabolic characteristics in cardiac maturation. Moreover, it highlights the potential use of the widely available clinical nuclear medicine tracers as a functional assay in stem cell research for improved generation of autologous cardiomyocyte-like cells for numerous biomedical applications. $$graphic_2DB40D06-6E63-488D-8364-18C77CFC178C$$