Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

Abstract

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in nonmatured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

NanoMEA: A Tool for High-Throughput, Electrophysiological Phenotyping of Patterned Excitable Cells

“…The application of ion-permeable nanotopographies to assay-ready MEA plates makes the core technology described here “assay agnostic,” meaning that identical substrates could be applied to any electrode-based assay and confer similar structural advantages on cultured cells. Consequentially, the described methods are applicable to other organ-on-chip devices and could be utilized to drive cardiac or neuron maturation within a range of preclinical, electrode-based screening assays for use in drug development or disease modeling. − This versatility greatly enhances the potential impact of the described technique and extends the observations for cardiac and neuronal cells described here to encompass a wide array of in vitro functional screening platforms.…”

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues

“…It was of interest to investigate the Ca 2+ transients of dystrophic EHTs, as previous studies have pointed to Ca 2+ dysregulation as a key phenotype of DMD presented in vitro. 22 – 25 , 28 – 31 To assess the extent of Ca 2+ dysregulation in DMD 263delG EHTs, the ratiometric Ca 2+ dye Fura-2 was used to assess both the kinetics of Ca 2+ transients as well as the relative cytosolic Ca 2+ levels under electrical field stimulation at 1.5 Hz. Comparing the ratiometric fluorescence intensities ( Figure 5(a) ) revealed significantly higher baseline levels of cytosolic Ca 2+ ( Figure 5(b) ) as well as higher peak levels ( Figure 5(c) ) but with a decreased amplitude of Ca 2+ flux in DMD 263delG EHTs ( Figure 5(d) ).…”

“…The contribution of Ca 2+ overload to the contractile deficiencies in dystrophic EHTs can be investigated through the use of Ca 2+ channel blockers or membrane sealants, as has been shown in previous in vitro studies. 23 , 24 Exon-skipping 26 , 64 and gene editing 29 , 65 – 67 have been shown to restore dystrophin expression in 2D hiPSC-CMs and limited studies have shown recovery of force of contraction in 3D engineered cardiac tissues, 25 , 52 but these studies have fallen short of describing a robust functional recovery in vitro. Through the studies described herein, we have made significant progress toward the development of a human-derived model of dystrophic cardiomyopathy.…”

High-Throughput Contractility Assay for Human Stem Cell-Derived Cardiomyocytes

“…Since adult cardiomyocytes are not available from humans and current techniques to mature hPSC-CMs are not yet able to achieve full adult-like structure, other single cell assays have been developed to quantify sarcomere structure. Higher throughput means of determining relative displacement of monolayers of cardiomyocytes includes correlationbased correction quantification (CCQ) 7 and FFT to directly monitor changes in sarcomere length 8 . There are additional methods that more directly quantify the forces applied by cardiomyocytes on their surroundings, such as micro-post platforms 9 and traction force microscopy 10 .…”

“…It is impressive that we now have tools that have the resolution to measure so many myofibrils in the cell. It would be exciting to combine this imaging technology with current cell patterning strategies to generate well aligned myocytes and/or other maturation techniques to obtain mature and aligned sarcomeres for analysis 7 . This will help to reduce the amount of noise generated in the data and be well suited to disease modeling and drug discovery studies.…”