Rapid pacing by circulating traveling waves improves maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring

Abstract

30Directed differentiation methods allow acquisition of high-purity cardiomyocytes (CMs) 31 differentiated from human induced pluripotent stem cells (hiPSCs); however, their 32 immaturity characteristic limits their application for drug screening and regenerative therapy. 33 The rapid electrical pacing of cardiomyocytes have been used for efficiently promoting the 34 maturation of cardiomyocytes, here we describe a simple device in modified culture plate on 35 which hiPSC-derived CMs (hiPSC-CMs) can form three-dimensional self-organized tissue 36 rings (SOTRs). Using calcium imaging, we show that within the ring, traveling waves (TWs) 37 of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 38 2 weeks, SOTRs with TW training showed matured features including structural organization, 39 increased cardiac-specific gene expression, enhanced Ca 2+ -handling properties, an increased 40 oxygen-consumption rate, and enhanced contractile force. We subsequently used a 41 mathematical model to interpret the origination, propagation, and long-term behavior of the 42 TWs within the SOTRs. This new idea for spontaneous hiPSC-CM maturation also has 43 potential for pacing the electrical excitable cells such as neuron and retina cells for various 44 applications. 45 46 47 48

Analysis of Circulating Waves in Tissue Rings derived from Human Induced Pluripotent Stem Cells

“…When we set the diameter to 3 mm, 1-, 2-, and 3-wave cases occurred. As the number of waves increased, their speeds decreased -1 wave propagated faster than 2 or 3 waves -and this trend was consistent in experiments 6 and simulations. Then, focusing on 1 wave in different diameter rings, we found that the propagation speed in the rings increased with the diameter both in simulations ( Fig.…”

“…Analytical stability criteria for the degree of cellular coupling. In the experiments, we observed that the number of waves decreased and the beating frequency increased with the increase of culture time 6 . We then investigated the potential causes for these phenomena.…”

“…Our previous data showed that cardiac myofilaments, trained by traveling waves, were arranged densely along the ring orientation, with the length of sarcomeres becoming significantly longer and the cardiac specific proteins, such like troponin T and α-actinin, being up-regulated. These experimental results indicated that TWs in SOTRs can promote maturation of hiPSC-CMs 6 .…”

“…1a). As indicated by the genetically-encoded calcium indicator GCaMP3, we found activation and looped propagation on the ring by using a fluorescence microscope equipped with a CCD camera as described previously 6 . GCaMP3 was excited at the wavelength from 450 nm to 490 nm, and the images were recorded with 8 × 8 binning of CCD pixels at 30 frames/s.…”

“…In previous studies 6 , we created a platform on which hiPSC-CMs self-organized into three-dimensional tissue rings (SOTR), where action potentials spontaneously originated and propagated in the form of traveling waves (TWs) (Fig. 1a).…”