Neuregulin-1β induces mature ventricular cardiac differentiation from induced pluripotent stem cells contributing to cardiac tissue repair

Abstract

Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1β (NRG-1β) treatment could enhance in vitro generation of mature "working-type" CMs from induced pluripotent stem (iPS) cells and assess the regenerative effects of these CMs on cardiac tissue after acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from α-MHC-GFP mice were derived and differentiated into CMs through NRG-1β and/or dimethyl sulfoxide (DMSO) treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, quantitative real-time polymerase chain reaction, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium control were injected into the peri-infarct region of hearts after coronary artery ligation, and functional and histology changes were assessed from 1 to 8 weeks post-transplantation. On differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1β and DMSO compared with DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1β and DMSO leads to efficient differentiation of iPS into ventricular-like cardiac cells with a higher degree of maturation, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.

FIG. 1.

Gene expression analysis of induced pluripotent stem-cardiomyocytes (iPS-CMs). (A) Unsupervised clustering of the samples: iPS-derived CMs (iPS-CMs) differentiated after treatment with dimethyl sulfoxide (DMSO) and/or neuregulin-1β (NRG-1β), neonatal CMs (NCMs), adult CMs (ACMs), and undifferentiated iPS cells (iPS). (B) Venn's diagram representing the differentially expressed genes (logFC >1) over parental iPS cells, in iPS-CMs and NCMs (left) and iPS-CMs and ACMs (right). (C) Biological function enrichment of the differentially expressed genes in cardiac categories (cardiogenesis and heart contraction) in iPS-CMs and CMs in comparison with parental iPS cells. (D) Heatmap and clustering of the differential gene expression observed between ACMs and NCMs in cardiac categories along with the iPS-CMs. Color images available online at www.liebertpub.com/scd

FIG. 2.

NRG-1β potentiates cardiac differentiation in iPS cells. (A) Differentiation of iPS-1 was induced with DMSO and/or NRG-1β. GFP⁺ clusters were detected at day 7 and 14 of differentiation, with larger GFP areas observed with combined treatment. Scale bars: 375 μm. (B) GFP⁺ areas were quantified at day 7 and 14 of differentiation. The percentage of GFP⁺ clusters was significantly higher when iPS cells were co-treated with DMSO and NRG-1β compared with either treatment alone (*P<0.05, **P<0.01, and ***P<0.001). Data are expressed as mean±standard deviation (SD). Color images available online at www.liebertpub.com/scd

FIG. 3.

Electrophysiological properties of iPS-CMs. (A) Phenotype of iPS-CMs. Cells were differentiated by treatment with DMSO and/or NRG-1β. The electrophysiological properties were analyzed at day 7 and 14 of differentiation. Spontaneous action potential (AP) recordings indicated that ventricular-like differentiation had occurred in all of the experimental groups. (B) Electrophysiological maturation of iPS-CMs. APD50, APD90, amplitude, maximal diastolic potential (MDP), V_max, and frequency parameters were measured at day 7 and 14. Measurements (n=10–14) were performed in several beating clusters from each experimental group. Even though the AP parameters of iPS-CMs were consistent with cultivated fetal CMs, more mature electrophysiological properties were associated with NRG-1β-treated CMs. Results are shown as mean±SD. *P<0.05, **P<0.01, and ***P<0.001 in comparison with DMSO.

FIG. 4.

Transplantation of iPS-CMs preserves cardiac function. Cardiac function was measured by echocardiography at 2, 30, and 60 days post-transplantation in control (medium) and treated animals (iPS-CMs). Left ventricular ejection fraction percentage (LVEF%) and fractional area change percentage (FAC%) are displayed (*P<0.05, between 2 and 60 days). Data are expressed as mean±SD.

FIG. 5.

Fate of iPS-CMs after transplantation. (A) Analysis of cell engraftment. Representative areas of injured myocardium are shown at 7, 14, 30, and 60 days after transplantation. GFP⁺ areas were observed at day 7 and 14, but no GFP⁺ signals were detected at longer time points (day 30 and 60). (B) Transplanted CMs were GFP⁺ [αMHC; (green), cardiac Troponin⁺ (cTNT; cyan), and Cx43⁺ (red)]. Connexin 43 (Cx-43) protein was distributed between the CMs, indicating the presence of gap junctions (electrical coupling). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). (C) Transmission electron microscopy of iPS-CMs at 7 days post-transplantation. Presence of GFP⁺ transplanted iPS-CMs was confirmed by electron microscopy (n=3). Organized and occasionally ramified myofibrils with Z-lines were detected throughout the cytoplasm (white arrows). Intercalated discs composed of desmosomes (black arrows) were detected between GFP⁺ iPS-CMs (red star), indicating mechanical coupling. Scale bars: (A) 50 μm; (B) 20 μm; (C) 2 μm (higher magnification: 1 μm). Color images available online at www.liebertpub.com/scd

FIG. 6.

Transplantation of iPS-CMs is associated with adequate tissue remodeling. Morphometric analysis was analyzed at 2 months after transplantation of either medium or iPS-CMs. Serial Sirius-red staining of sections revealed significantly reduced scar sizes in the left ventricles of the iPS-CM-treated hearts compared with media-treated hearts (A–C). Data show the percentage of infarcted area (IA) versus the total left ventricular area (LVA). The degree of fibrosis was measured in the peri-infarct zone and was significantly lower with transplantation of iPS-CMs compared with the control (D–F). Capillary and arterioles/arteries densities were determined through quantification of caveolin-1⁺ (capillaries/mm²; 5–15 μm diameter) (G–I) and α-smooth muscle actin (SMA⁺) vessels (μm²/mm²) (J–L) in the peri-infarct area. Data are expressed as mean±SD (*P<0.05). Scale bars: (A, B) 500 μm; (D, E) 50 μm; (G, H, J, K) 100 μm. Color images available online at www.liebertpub.com/scd