Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.

FIG. 1.

Characterization of cardiac ECM from decellularized bovine fetal and adult heart tissue. Decellularized adult bovine cardiac tissue has a more prominent fibrillar structure than fetal cardiac tissue. (A, C, E, and G) Fetal heart tissue. (B, D, F, and H) Adult heart tissue. (A, B) Control tissue. (C–H) Decellularized tissue. (E, F) Elastin fibers visualized by autofluorescence at 860 nm. (G, H) Collagen fibers visualized by second-harmonic generation at 740 nm. (I) Quantification of the number of collagen fibers comparing fetal and adult decellularized cardiac tissue. (J) Collagen fiber width comparing fetal and adult decellularized cardiac tissue. Results are expressed as mean ± standard error (n = 3; *p < 0.05, **p < 0.01, Student's t-test). “#” refers to number of collagen fibers per field of view (FOV). ECM, extracellular matrix. Color images available online at www.liebertpub.com/tea

FIG. 2.

3D adult cardiac ECM increases iPSC-derived CM expression of structural and functional cardiac genes. Gene expression was measured using qRT-PCR and expression normalized to 2D fetal cardiac ECM. Cardiac genes measured were JCN, CACNA1C/CaV1.2, Cx43, CASQ2, NCX1, and MYL2. (A) Gene expression in iPSC-derived CMs cultured in (3D) or on (2D) fetal cardiac ECM (*p > 0.01, **p > 0.001, Student's t-test). (B) Gene expression in iPSC-derived CMs cultured in (3D) or on (2D) adult cardiac ECM. (C, D) Data from (A, B) replotted to aid direct comparison of gene expression in iPSC-derived CMs cultured on adult and fetal 2D cardiac ECMs (C) and 3D cardiac ECM (D). Unless otherwise indicated, results are expressed as mean ± standard error. (n = 3; ^δp > 0.05, ^δδp > 0.01, Split-Unit ANOVA). 2D, two-dimensional; 3D, three-dimensional; CACNA1C/CaV1.2, L-type voltage-dependent calcium channel; CASQ2, calsequestrin 2; Cx43/GJA1, connexin-43; iPSC, induced pluripotent stem cell; JCN, junctin; MYL2, myosin light chain 2; NCX1, sodium–calcium exchanger 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

FIG. 3.

3D adult cardiac ECM increases iPSC-derived CM protein expression of structural and calcium-handling proteins. iPSC-derived CMs express sarcomere and calcium-handling proteins. (A) Flow cytometry analysis for cTNT(+) cells after 21 days of differentiation. (B) Immunofluorescent staining for MYL2 and CaV1.2 in iPSC-derived CMs cultured in 2D and 3D cardiac ECMs. (Right panels) MYL2 staining. (Left panels) CaV1.2 staining. Scale bar is 50 μm. Color images available online at www.liebertpub.com/tea

FIG. 4.

3D adult cardiac ECM increases calcium signaling in iPSC-derived CMs. A GCaMP reporter was used to visualize calcium transients in iPSC-derived CMs after 7 days in 2D and 3D adult cardiac ECMs. (A–E) iPSC-derived CMs cultured in 3D as single cells displayed decreased calcium signaling and kinetics. (F–J) iPSC-derived CMs cultured in 3D as aggregates displayed an increase in calcium signaling and kinetics compared with 2D. (A) Beat rate of iPSC-derived CMs cultured in 2D and 3D adult/fetal cardiac ECMs. (B, G) Amplitude. (C, H) Max Upslope. (D, I) Max Downslope. (E, J) Time to 50% decay. (F) Representative calcium wave transient of iPSC-derived CM culture in 2D and 3D adult cardiac ECMs. Results are expressed as mean ± standard error (n = 4–12; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test).

FIG. 5.

3D adult cardiac ECM increases iPSC-derived CM response to drugs. iPSC-derived CMs cultured in 3D were more responsive to β-adrenergic stimuli and caffeine than cells in 2D cultures. (A) The effect of isoproterenol and propranolol on the beat rate of iPSC-derived CMs. (B) A representative fluorescent signal of the iPSC-derived CM calcium transient in response to 20 mM caffeine cultured in 2D and 3D adult cardiac ECMs. (C) The time to maximum fluorescent output (F_max) was measured after the addition of caffeine. (D) The velocity of calcium release F/time. Results are expressed as mean ± standard error (n = 13–17; **p < 0.01 ***p < 0.001, Student's t-test). Color images available online at www.liebertpub.com/tea