Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses

Abstract

Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (μHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

Figure 1. Simple stencil-based strategy to produce Micro-Heart Muscle.

(A) Schematic describing strategy to produce Micro-Heart Muscle (μHM) arrays. Through-holes in stencils are seeded with a combination of iPS-CM (red) and fibroblasts (yellow), and the geometry of these regions generates a uniaxially-stressed, substrate-anchored tissue. (B) Representative images of iPS-CM and isogenic fibroblasts combined and seeded into (left) or square-shaped (right) through-holes. Red vectors quantify direction and magnitude of contractile motion during maximum contraction velocity (peak contractility). (C) Quantification of the direction of motion of all motion vectors, for tissue formed within rectangular through-holes with constant area (4 × 10⁴ μm²) but varying width (*p < 0.05 compared to “no-pattern” condition). Direction was quantified via the percentage of vectors that were longitudinal (blue) versus transverse (red) to the long-axis of the rectangular through-hole. (D) Representative time-course images depicting assembly of substrate-anchored μHM, with cell-adhesive “knobs” connected by a shaft, and (E) quantification of μHM integrity, as measured by the relative area occupied by tissue within the cell-adhesive region of dogbone stencil patterns, for μHM formed with knobs of varying geometry. (F) Quantification of the percent of motion that is longitudinal versus transverse in the shaft and knob regions of μHM. (G) Representative whole-mount immunofluorescence staining for sarcomeric α-actinin (green, with Hoechst nuclear counterstain, blue) in a 2-week-old μHM. (H–K) Representative scanning electron micrographs depicting a substrate-anchored μHM, indicating assembly of fiber structures on the micron and sub-micron scales. Error bars: SEM, n = 5–6 (*p < 0.05, ***p < 10⁻⁵). Scale bars: B: 400 μm (top left); 200 μm (top right); 100 μm (bottom); D: 500 μm; G: 50 μm (inset: 10 μm); H: 100 μm; I: 20 μm; J: 10 μm; K: 200nm. Error bars are SEM, n = 5–6).

Figure 2. Cardiomyocyte morphology and distribution within Micro-Heart Muscle.

(A–D) Representative confocal cross-sections of μHM assessed by whole-mount staining for (A) filamentous actin and nuclei (F-actin, green, and propidium iodide, red), (B) Myosin Light Chain 2v (MLC2V, green), and Myosin Light Chain 2a (MLC2A, Blue), (C) Cardiac Troponin I-C (TNNI3, green) and nuclei (propidium iodide, red), and (D) for sarcomeric α-Actinin (ACTN2, green), phospho-Connexin 43 (pCx43, red) and Vimentin (blue). (E) Representative confocal cross-section of an adult mouse ventricle stained for sarcomeric α-Actinin (Actn2, green) and pCx43 (red). (F–H) Analysis of the distribution of stromal cells (Vimentin, blue) and cardiomyocytes in the μHM (F) Cardiac Troponin T, TNNT2, green; (G–H) ACTN2, green) in (F) low magnification and (G–H) high magnification confocal cross-sections of μHM. (I) Representative images of iPS-CM harboring doxycycline induced TetO-ACTN2-mKate2 which were co-cultured with isogenic stromal cells either within monolayers (left) or μHM (right). Note cardiomyocytes form sarcomeres in both conditions, but cells appear much more elongated within μHM. (J) Quantification of the cellular aspect ratio of ACTN2-mKate2-positive iPS-CM within either μHM or confluent monolayers (**p < 10⁻⁴, 2-way t-test). Data points with the same aspect ratio value are stacked horizontally for easier viewing. Scale bars: (A–E) 10 μm; (G–H) 20 μm; (F,I) 50 μm.

Figure 3. Physiology of iPS-CM within Micro-Heart Muscle Arrays.

(A) Representative μHM image; two adjacent μHM are noted as “1” and “2”. (B) Tracings of the root-mean-squared beat speed due to spontaneous contractility the two adjacent μHM, noted in (A). Note tracings indicate that although the μHM have similar rates of beating (a doublet of peaks denotes one contraction-relaxation cycle), they are not beating in a correlated manner. (C,D) Representative (C) image and (D) tracings of calcium flux (GCaMP6 levels) in two adjacent μHM, indicating that the individual tissues have independent calcium flux. (E,F) Chronotropic response to a 10 μM pulse of isoproterenol of iPS-CM co-cultured with isogenic stromal cells within (E) monolayers or (F) μHM arrays. Note μHM arrays formed with different batches of iPS-CM and isogenic EB-stromal cells (derived and purified independently) are colored differently in (F). (G) IC₅₀ analysis for Verapamil, as monitored via contractility (maximum contraction velocity, normalized to maximum contraction velocity in the same tissue before drug treatment), in for iPS-CM and fibroblasts cultured in monolayer (open black squares) or μHM (formed from three different batches of iPS-CM and isogenic EB-stromal cells; solid black diamonds, blue circles and red triangles). (H) Representative tracing of (H) calcium flux (GCaMP6f fluorescence) or (I) radial contraction velocity of the shaft region in 2-week μHM either without field pacing or pacing up to 2 Hz. Note in I, tissue was paced after removing the stencil. Error bars: SEM, n = 5 (monolayer), or 4–10 (μHM).

Figure 4. Organ Bath Physiology of Micro-Heart Muscles after Removal from Array Format.

(A) Schematic (top) and representative image (bottom) of a μHM mounted onto hooks attached to a tether on one end and a strain gauge and micro-manipulator on the other end. (B) Force over time of a mounted 2 week μHM (0.1 mN baseline force) either beating spontaneously, or subjected to field pacing at 1–4 Hz. (C) Representative Frank-Starling analysis of a μHM. Tissue was stretched by 50 μM at regular intervals, and the resultant baseline and twitch force were recorded, demonstrating an increase in twitch force. (D) Quantification of the Frank-Starling response in mounted μHM. Tissue length was defined as 1 at the length that yielded maximum twitch-force, which plateaued thereafter. (E) Quantification of calcium dose response (increasing twitch force) in μHM (EC₅₀: 1 mM). (F) Twitch force of μHM within five minutes of treatment with increasing doses of isoproterenol. Error bars: SD, n = 3 (pooled from two independent batches of iPS-CM and EB-stromal cells). Note for normalized tissue length of 1.1, n = 1.