Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes

Abstract

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Figure 1. Structural properties of human cardiac tissue patches

A) Representative 2-week old cardiac tissue patch anchored within a Velcro frame. B-C) Staggered elliptical pores within the patch (B) are surrounded by densely packed and aligned cells (C). D) hESC-CMs in 2-week old cardiac tissue patches are aligned and show cross-striated patterning of cardiac Troponin T (cTnT). Fibroblasts positive for vimentin (Vim) are interspersed among hESC-CMs. E) hESC-CMs also exhibit cross-striated pattern of myosin heavy chain (MHC) and sarcomeric α-actinin (SAA). F) Cardiac patches show evidence of mechanical coupling (N-cadherin) between hESC-CMs as well as presence of smooth muscle cells (SM22α). Data shown for patches made with 70% hESC-CMs.

Figure 2. hESC-CMs in 3D patches exhibit longer sarcomeres than in 2D monolayers

A) Representative immunostainings of hESC-CMs in 2-week old tissue patch coupled by connexin-43 gap junctions. B) hESC-CMs in 2-week old monolayers. C) Histogram distribution of sarcomere lengths in 2-week old patches and monolayers made with 48-65% hESC-CMs. Note increased sarcomere length in patches vs. monolayers. D) Median and quartile sarcomere lengths in monolayers (n=58 hESC-CMs) and patches (n=106 hESC-CMs). *p<0.0001.

Figure 3. Quantitative gene expression during patch and monolayer culture

A-C) qPCR readout of pluripotency (OCT4 and NANOG) and cardiac progenitor (ISL1 and GATA4) genes in undifferentiated hESCs (ES), beating cell clusters (PM0), and cardiac patches at 4 days (P4D), 1 week (P1W), and 2 weeks (P2W) of culture. D-F) qPCR readout of contractile function genes: MHC, myosin heavy chain; cTnT, cardiac Troponin T; MLC2v/a, Myosin light chain 2 ventricular/atrial. G-I) qPCR readout of electrical function (Nav1.5, fast Na⁺ channel; Kir2.1, inward rectifier K⁺ channel; Kv4.3, transient outward K⁺ channel; Cav1.2, L-type Ca²⁺ channel) and excitation-contraction (SERCA2, SR Ca²⁺-ATPase; CASQ2, calsequestrin) genes in patches and monolayers of the same age (M1W and M2W, 1 and 2 week monolayer) and hESC-CM purity (60-65%). All data except F are shown relative to PM0 group. n = 2–4 biological × 3 technical replicates. p<0.05 from: *, PM0; **, all other groups; †, ES; #, another group.

Figure 4. Action potential propagation in cardiac patches and monolayers

A) Representative optically mapped action potential traces in a 2-week old cardiac patch at various pacing rates. B,C) Representative isochrone maps during 1 Hz point stimulation of a 2-week old cardiac patch (B) and monolayer (C). D-F) Conduction velocity (D), action potential duration (E), and maximum capture rate (F) during point stimulation of cardiac patches and monolayers as a function of hESC-CM purity. †p<0.05 between patches and monolayers (48% < hESC-CM purity < 65%.)

Figure 5. Contractile properties of cardiac tissue patches

A) Representative active (contractile) force traces during progressive stretch of an electrically stimulated (1Hz) 2-week cardiac patch. B) Corresponding active and passive force-length relationships in cardiac patches made with 57%-65% hESC-CMs (n=5). C) Maximum contractile force (at 1 Hz stimulation) in 2-week patches as a function of hESC-CM purity. D) Contractile force per input hESC-CM as a function of cardiomyocyte purity; p<0.03 for linear trend.

Figure 6. Inotropic response of cardiac tissue patches to beta-adrenergic stimulation

A) Representative active force traces in a tissue patch made with 55% hESC-CMs during application of increasing doses of isoproterenol (measured at 10% stretch, 1 Hz stimulation, and 0.9 mM extracellular [Ca²⁺]). B-C) Contractile force amplitude (B) and twitch kinetics (C) as a function of isoproterenol concentration (n=4).