Three-dimensional filamentous human diseased cardiac tissue model

Abstract

A human in vitro cardiac tissue model would be a significant advancement for understanding, studying, and developing new strategies for treating cardiac arrhythmias and related cardiovascular diseases. We developed an in vitro model of three-dimensional (3D) human cardiac tissue by populating synthetic filamentous matrices with cardiomyocytes derived from healthy wild-type volunteer (WT) and patient-specific long QT syndrome type 3 (LQT3) induced pluripotent stem cells (iPS-CMs) to mimic the condensed and aligned human ventricular myocardium. Using such a highly controllable cardiac model, we studied the contractility malfunctions associated with the electrophysiological consequences of LQT3 and their response to a panel of drugs. By varying the stiffness of filamentous matrices, LQT3 iPS-CMs exhibited different level of contractility abnormality and susceptibility to drug-induced cardiotoxicity.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales

“…As in previous studies, efficient differentiations yielded sheets of cardiomyocytes wherein at least 70% of cells were positive for cardiac troponin T by FACS analysis 15 (Data not shown). Optical flow analysis applied to these sheets yielded similar results to those previously reported for sheets of neonatal or human embryonic stem cell-derived cardiomyocytes.…”

“…Therefore, this system may be suitable for comparing the behavior, drug response, and maturation of isolated iPS-CM and iPS-CM grown in more in vivo-like microenvironments. 15,25,26 To this end, the enabling nature of this software was demonstrated in a previous study, in which long QT syndrome type 3 patient-specific iPSCs were differentiated into iPS-CM and cultured on Response surface analysis of the combined effects of isoproterenol dose and spontaneous (initial) beat rate on iPS-CM chronotropic (beat rate) response to isoproterenol dosing. Only the spontaneous initial beat rate had significant effects on the fold increase in beat rate (Pearson correlation analysis).…”

“…Using the modified GiWi differentiation protocols (above), we achieved differentiation efficiencies of above 70% (based on flow cytometry analysis of cardiac Troponin T 15 ), and only wells that had visibly beating sheets were used for tracking analysis. For replating, iPS-CM on days 15-18 of differentiation were retrieved by digestion for 10-15 min in 0.25% trypsin, which was quenched in embryoid body media 20 (EB20; Knockout Dulbecco's Modified Eagle's Medium containing 20% characterized fetal bovine serum, 1 mM nonessential amino acids, 1 mM lglutamine, 0.1 mM b-mercaptoethanol).…”

“…8,10 These techniques require the user to specify regions of interest to monitor, which introduces inherent bias. 8,11 To avoid this bias, a series of more robust methods to detect motion, collectively referred to as optical flow, 12 are now used to analyze primary 13,14 and pluripotent stem cell-derived 9,15 cardiomyocytes. In the field of computer vision, optical flow describes the motion of pixels from one frame to the next in a two-dimensional (2D) video.…”

“…[12][13][14][15] However, because of technical obstacles, many users have not adopted this unbiased approach for analyzing cardiomyocyte contractility. First, analyzing videos of single or sparse cardiomyocytes is difficult because their contraction speed is often obscured by nearby Brownian motion or camera noise.…”