In vivo grafting of large engineered heart tissue patches for cardiac repair

Abstract

Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.

Keywords: Arrhythmias; Cardiology; Cardiovascular disease; Human stem cells; Stem cells.

Figure 1. In vitro characterization of EHTs.

(A) Equipment used to make silicone mold and to generate EHTs. (B) Macroscopic image of EHT just prior to grafting. (C) Immunostained EHT (troponin T abbreviated as TNNT2; fibroblasts; vimentin) 28 days after generation. Scale bar: 1000 μm. (D) Higher magnification image of C (TNNT2, fibroblasts, vimentin). Scale bar: 20 μm. (E) Graph of late EHT calcium transient (paced 1 Hz) and calcium transient differences between early and late EHTs (early, <2 weeks; late, >4 weeks). (F) Troponin T cell alignment data (n = 4 early; n = 8 late) and contraction kinetics over time (n = 6). Data are presented as mean ± SEM, and 2-way ANOVA and unpaired Student’s t test were the statistical methods used. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2. EHT grafting on controls.

(A) Macroscopic image of EHT attached to epicardial surface 2 weeks postexplant. Red dotted lines show the EHT and host border zone. (B) Axial sections from 4 different animals postexplant (EHT outlined by blue dotted lines); scale bar: 1500 μm. (C) Troponin T (TNNT2) and hematoxylin staining of host/EHT; scale bar upper panel: 300 μm. (D) Ku80 and hematoxylin staining of host/EHT; scale bar upper panel: 300 μm. (E) Troponin retention at stratified time points relative to day 0. Data presented as mean ± SEM and 1-way ANOVA used to compare groups. ***P < 0.001.

Figure 3. EHTs are vascularized by the host.

(A) CD31 staining of EHT/host with higher magnification image (right panel); scale bar left panel: 300 μm; right panel: 100 μm. (B) CD31 (left panel) vessels and Ku80 nuclei stain of serial section; scale bar: 100 μm; red dotted line shows the heart; yellow dotted line shows the EHT border. (C) Graph of EHT capillary density over time. (D) Bar chart of capillary density of proximal and distal parts of EHT at serial time points (week 1 n = 3, week 2 n = 6, week 3 n = 2, and week 4 n = 7). Data presented as mean ± SEM and 1-way ANOVA used to compare groups. *P < 0.05, **P < 0.01.

Figure 4. EHTs improved ventricular function when grafted onto infarcted hearts.

(A) Fractional area change (FAC). **P < 0.01. (B) Anterior wall thickness (2-way ANOVA). (C) Quantification of Sirius red infarct staining of rabbit heart sections (n = 7 sham; n = 7 EHT: sham: 1 week n = 1; 4 weeks n = 6; EHT 1 week n = 1; 2 weeks n = 2; 4 weeks n = 4). *P < 0.02, Mann-Whitney t test. (D) Correlation of FAC measurements between 2 echocardiographers blinded to treatment allocation (n = 42 measurements). Data presented as mean ± SEM (A and B); data presented as median with 95% confidence intervals (C).

Figure 5. EHT optical mapping.

(A) Macroscopic image of EHT/rabbit heart explanted and mapping 1 week postgrafting. (B) Troponin T staining (brown area) of animal in A, host/EHT separated by red dotted line; scale bar: 200 μm. (C) Fluorescence map of animal in A; (D) calcium transients of C; numbers correspond to numbers in C. (E) Fluorescence map of animal 2 weeks postgrafting. (F) Respective calcium transients of E.

Figure 6. Arrhythmogenic potential of EHTs.

(A) Representative ECG traces obtained from ex vivo programmed electrical stimulation protocols. (B) Arrhythmia inducibility scores (ex vivo) and in vivo ventricular ectopics (VE) burden (%) recordings obtained from in vivo telemetry. (C) Representative traces of ventricular tachycardia, VE, and junctional rhythm obtained from in vivo telemetry recordings. Data presented as median and 95% CI and Kruskal-Wallis test used to compare groups. *P < 0.05.

Figure 7. Timeline for in vivo experiments.

In vivo grafting protocol.