N-cadherin overexpression enhances the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes in infarcted mouse hearts

Xi Lou; Meng Zhao; Chengming Fan; Vladimir G Fast; Mani T Valarmathi; Wuqiang Zhu; Jianyi Zhang

doi:10.1093/cvr/cvz179

. 2019 Jul 26;116(3):671–685. doi: 10.1093/cvr/cvz179

N-cadherin overexpression enhances the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes in infarcted mouse hearts

Xi Lou ¹, Meng Zhao ¹, Chengming Fan ¹, Vladimir G Fast ¹, Mani T Valarmathi ¹, Wuqiang Zhu ¹, Jianyi Zhang ^1,^✉

PMCID: PMC8204485 PMID: 31350544

Abstract

Aims

In regenerative medicine, cellular cardiomyoplasty is one of the promising options for treating myocardial infarction (MI); however, the efficacy of such treatment has shown to be limited due to poor survival and/or functional integration of implanted cells. Within the heart, the adhesion between cardiac myocytes (CMs) is mediated by N-cadherin (CDH2) and is critical for the heart to function as an electromechanical syncytium. In this study, we have investigated whether the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) can be enhanced through CDH2 overexpression.

Methods and results

CDH2-hiPSC-CMs and control wild-type (WT)-hiPSC-CMs were cultured in myogenic differentiation medium for 28 days. Using a mouse MI model, the cell survival/engraftment rate, infarct size, and cardiac functions were evaluated post-MI, at Day 7 or Day 28. In vitro, conduction velocities were significantly greater in CDH2-hiPSC-CMs than in WT-hiPSC-CMs. While, in vivo, measurements of cardiac functions: left ventricular (LV) ejection fraction, reduction in infarct size, and the cell engraftment rate were significantly higher in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group. Mechanistically, paracrine activation of ERK signal transduction pathway by CDH2-hiPSC-CMs, significantly induced neo-vasculogenesis, resulting in a higher survival of implanted cells.

Conclusion

Collectively, these data suggest that CDH2 overexpression enhances not only the survival/engraftment of cultured CDH2-hiPSC-CMs, but also the functional integration of these cells, consequently, the augmentation of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts.

Keywords: N-cadherin, Human-induced pluripotent stem cells, Cardiac myocytes, Myocardial infarction, Cardiac regeneration, Electro-mechanical syncytium

Graphical Abstract

1. Introduction

In cardiovascular regenerative and reparative medicine, the most fundamental problem facing cardiac therapy is to repair and/or regenerate the damaged myocardium. Restricted myocardial regeneration after tissue damage and shortage of donor organs for cardiac transplantation are the principal constraints of conventional therapy.¹ As a result, at present, in regenerative medicine, cellular cardiomyoplasty is one of the promising therapeutic options, especially for the maintenance and repair of various cardiac disorders, such as post-infarction left ventricular remodelling, dilated cardiomyopathies, and concentric hypertrophy.^2–10

Preclinical and clinical studies have shown that whole marrow isolates and/or cultured somatic-derived stem and/or progenitor cells, e.g. mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), as well as induced pluripotent stem cells (iPSCs), may possibly contribute to myocardial repair in vivo and alleviate cardiac symptoms, although the mechanisms for this remain obscure.⁴ ^, ^11–13 These cells have also been grafted onto ischaemic heart tissue, but their extent of functional integration remains unresolved.⁴ ^, ¹¹ ^, ¹⁴ ^, ¹⁵ Collective evidence demonstrates that exogenous stem cells, irrespective of the modes of administration into cardiac lesions, generated relatively few neo-cardiomyocytes displaying an immature phenotype.¹⁶ Besides, these cells may well promote revascularization after myocardial injury.³ Taken together these facts suggest that, strategies aiming at the improvement of cellular cardiomyoplasty to repair and/or regenerate regions of damaged myocardium are of critical importance.

Until now, the use of somatic tissue-derived stem cells in the stimulation of cardiac regeneration is still in its early stages. Unlike human embryonic stem cells, the advent of human-induced pluripotent stem cells (hiPSCs), created a way to generate autologous source of cardiac myocytes (CMs).⁴ Now, a large number of spontaneously beating CMs can consistently be derived from hiPSCs using chemically defined protocols.¹⁷ ^, ¹⁸ Despite the progress in directed differentiation, the hiPSC-derived neo-myocytes remained phenotypically immature.¹⁴ ^, ¹⁵ ^, ^19–21 Therefore, at present, the most important goal in cardiac therapy is the quest, not only improve cell survival and engraftment but also acquire a mature phenotype resulting in functional integration of cells at the site of implantation, and ultimately to recreate normal myocardial architecture and anisotropy.²²

N-cadherin, also known as neural cadherin (NCAD) or cadherin 2 (CDH2), is a transmembrane, homophilic glycoprotein. CDH2 is a one of the classical calcium-dependent cell adhesion proteins, involves in maintaining the physical association between cells, as disruption of them causes loosening of cell–cell contacts leading to disorganization of tissue architecture. CDH2 is widely expressed in multiple tissues, such as neuronal, cardiac, and other organs.²³ In cardiac muscle, CDH2 is essential for maintaining the structural and functional integrity of CMs, and CDH2 is an integral component of adherens junction (AJ) residing at intercalated discs, which facilitate mechanical and electrical coupling between adjacent CMs. The loss of CDH2 in myocytes could result in disrupted assembly of intercalated discs, decreased connexin 43 expression, dilated cardiomyopathy, arrhythmias, and impaired cardiac functions.^24–27 Besides, CDH2 has also been shown to affect a wide range of signalling networks, including cell proliferation, differentiation, apoptosis, and angiogenesis.²⁸ ^, ²⁹ Thus, CDH2 is associated with many molecules that regulate its function and is involved in a plethora of processes, including cell–cell adhesion, differentiation, migration, invasion, embryogenesis, and signal transduction.²³

hiPSC-based therapy for improving cardiac function during acute myocardial infarction (MI), so far, has been shown to be safe and has resulted in modest benefits on cardiac function.⁴ ^, ³⁰ Recently, some newer therapeutic strategies have, however, been explored for augmenting and/or promoting the effects of cell-based therapy, one of them is genetic enhancement of stem cells.⁵ ^, ⁶ ^, ⁹ As a result, combining stem cell therapy with gene therapy is a viable strategy for the prevention and treatment of cardiac dysfunctions.³¹ Therefore, in this study, using mouse MI model, we investigated whether the engraftment, electrical connectivity, and reparative potency of hiPSC-derived CMs can be enhanced through CDH2 overexpression, resulting in improved cardiac function post-MI.

2. Methods

All animal procedures were performed in accordance with the guidelines for animal experimentation set forth and approved by Institutional Animal Care and Use Committee (IACUC, APN 20502), School of Engineering, University of Alabama at Birmingham; and conformed to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011).

2.1 WT-hiPSC culture: expansion and maintenance

Wild-type hiPSCs (WT-hiPSCs) carrying an engineered luciferin gene, were expanded and subcultured according to previously published method (for details, see the Supplementary material online).^32–34

2.2 CDH2-hiPSCs culture: generation, expansion, and maintenance

Using CDH2 plasmid (Addgene No. 38153), CDH2 insert was generated by PCR amplification with a pair of oligos (forward: 5′-tcctatctcccccataagagtttgagtcgactgatcaaaaaccggtatgtgccggatagcgggagcgct-3′, reverse: 5′-ctgaagcttttctgtaatttacttatcgggaggaatcggtggcgcgccgtcatcacctccaccatacatgt-3′) (Invitrogen) (for details, see the Supplementary material online).

2.3 Cardiomyogenic differentiation: generation of WT-hiPSC-CMs and CDH2-hiPSC-CMs

WT-hiPSCs or CDH2-hiPSCs that were maintained and expanded in mTESR™1 medium, until 80–90% confluent, were subjected to cardiomyogenic differentiation using GiWi protocol, as described elsewhere (for details, see the Supplementary material online).¹⁷ ^, ³² ^, ³⁵ ^, ³⁶

2.4 Purification and metabolic selection of WT-hiPSC-CMs and CDH2-hiPSC-CMs

To prepare purified CMs for all of our experimental setups, the differentiated cells that were maintained in RPMI/B27 basal medium was switched to, modified glucose-free RPMI medium (no glucose, no pyruvate; Invitrogen) supplemented with 4 mM lactate and B27, and cultured for 4–7 days.³⁴ ^, ³⁷

2.5 Phenotypic characterization of WT-hiPSC-CMs and CDH2-hiPSC-CMs by single-colour flow cytometry

To determine the purity of hiPSC-derived CMs (i.e. WT-hiPSC-CMs or CDH2-hiPSC-CMs) among other differentiated cells, quantitative analysis for the cardiac-specific marker, such as human cTnT was performed on cells by single-colour flow cytometry using a BD™ FACSCalibur™ flow cytometer (Beckman, Dickinson and Company) as described previously.³⁸

2.6 Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)

Total cellular RNA was extracted with RNeasy Plus Mini Kit (Qiagen) as per manufacturer’s instructions (for details, see the Supplementary material online). Real-time PCR conditions were optimized as previously described (for details, see the Supplementary material online).³⁷

2.7 Optical mapping of action potentials (APs)

Action potentials (APs) and activation spread were mapped optically in monolayer cultures of WT-hiPSC-CMs and CDH2-hiPSC-CMs (for details, see the Supplementary material online).

2.8 Mouse myocardial infarction model: WT-hiPSC-CMs and CDH2-hiPSC-CMs implantation

Surgical induction of MI was performed on eight to ten weeks old NOD/SCID gamma mice [NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (005557); The Jackson Laboratory]. In brief, mice were intubated and connected to ventilator, while breathing 2% isofluorane USP (Fluriso™, VetOne^®) for maintenance of anaesthesia. The heart was exposed through left-sided thoracotomy, and MI was induced by a permanent ligation of the left anterior descending coronary artery (LAD) with a non-absorbable suture. Next, WT-hiPSC-CMs or CDH2-hiPSC-CMs (after 28 days of in vitro differentiation) were used for cell implantation purposes. Freshly harvested cells, either WT-hiPSC-CMs or CDH2-hiPSC-CMs (3 × 10⁵ CMs/mouse) suspended in Dulbecco’s phosphate buffered saline (DPBS), pH 7.4, were delivered into the infarct and border zones by means of three intra-myocardial injections (equal volume, 1 × 10⁵ CMs/site). For MI only group, cell implantation was not performed. For sham group, suture was passed around LAD without ligation. Finally, the open chest was closed, and buprenorphine hydrochloride (0.1 mg/kg, Buprenex^®, Reckitt Benckiser Pharmaceuticals Inc.) was given as intraperitoneal (IP) injections for every 12 h, up to 3 days post-surgery; and carprofen (5 mg/kg, Rimadyl^®, Zoetis) was given as IP injections for every 12 h, for 1 day post-surgery. Each group contained seven to nine animals.

2.9 Echocardiography

Post-MI (on Day 7 and Day 28), the WT-hiPSC-CMs MI and CDH2-hiPSC-CMs MI treated animals were subjected to echocardiographic imaging as previously described³⁹ (for details, see the Supplementary material online).

2.10 Determination of infarct size

Following the coronary occlusion, groups of seven to nine (post-MI Day 7 or Day 28) mice were reanaesthetized and killed by excision of the heart. The infarct size was evaluated as described previously⁴ ^, ⁴⁰ (for details, see the Supplementary material online).

2.11 Immunostaining and fluorescence microscopy

The whole animal hearts were collected at Day 7 or 28 and 10 µm serial cryosections were processed according to previously described protocols (for details, see the Supplementary material online).³⁷

2.12 Engraftment rate of WT-hiPSC-CMs and CDH2-hiPSC-CMs

Engraftment rate of implanted CMs, viz., WT-hiPSC-CMs or CDH2-hiPSC-CMs, were not only evaluated by histological assessment for implanted cells but also evaluated by bioluminescence imaging (BLI) and assessment, since the original WT-hiPSC cell line was genetically engineered to express the luciferase gene. Histological examination of cells was performed on cells that expressed simultaneously both human-specific cardiac troponin T (hcTnT) and human-specific nucleolin antigen (hNCL) (for details, see the Supplementary material online).

2.13 Hypoxic treatment of WT-hiPSC-CMs and CDH2-hiPSC-CMs

WT-hiPSC-CMs or CDH2-hiPSC-CMs were cultured in serum-free medium under hypoxic conditions (1% O₂, 5% CO₂, and 94% N₂) for 12, 24, or 48 h. Similarly, WT-hiPSC-CMs or CDH2-hiPSC-CMs were cultured under normoxic conditions for 12, 24, or 48 h, served as controls. The cultures (WT-hiPSC-CMs or CDH2-hiPSC-CMs) were terminated at these regular intervals, and the collected samples were subjected to terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay.

2.14 Detection of apoptotic cells by TUNEL assay

The in situ cell death detection kit (fluorescein, Roche) was used for the detection and quantification of apoptosis at the single-cell level using a fluorescence microscope. After incubation, culture samples (hypoxic WT-hiPSC-CMs/CDH2-hiPSC-CMs; normoxic WT-hiPSC-CMs/CDH2-hiPSC-CMs) were collected at 12, 24, or 48 h, rinsed twice in DPBS, pH 7.4, and fixed in 4% PFA at room temperature for 30 min. The labelling reactions with TUNEL reaction mixture were carried out as described by manufacturer’s instruction.

2.15 Quantification of angiogenesis

Angiogenic response of the border zone of infarcted myocardium, such as vessel density, was evaluated through immunostaining and fluorescence microscopy.

2.16 Western blotting

Lysis and extraction of total cellular proteins were carried out using M-PER^® mammalian protein extraction reagent (Thermo Fisher Scientific), consisting of a cocktail of protease- and phosphatase-inhibitors (Thermo Fisher Scientific), as per the manufacturer’s instructions (for details, see the Supplementary material online).

2.17 Statistical analysis

The data were represented as mean ± standard error of the mean (mean ± SEM). For all the experimental data, the differences among various groups were determined by applying one-way, two-way analysis of variance (ANOVA), and/or Student’s t-test. Likewise, other acquired quantitative data, such as image (morphometric) analysis, the number of replicates refers to the number of analysed images from five to eight independent experiments (n = 5–9), whereas, in the case of animal studies, the number of replicates refers to the number of analysed animals, 5–9 in each group (n = 5–9). In all cases, P < 0.05 were considered statistically significant.

3. Results

3.1 Phenotypic characterization of WT-hiPSC-CMs and CDH2-hiPSC-CMs

Since the maturation and differentiation, as well as the purity of hiPSC-derived CMs are critical for therapeutic purposes, we have analysed the differentiating myocytes for various significant cardiac-specific markers, before they could potentially be used in all of our experimental setups. Immunophenotyping and qualitative evaluation of Day 28 WT-hiPSC-CM and CDH2-hiPSC-CM by fluorescent microscopy revealed the expression pattern of human cardiac-specific regulatory protein, troponin T (hcTnT) and the cell adhesion protein, cadherin 2 (CDH2) (Figure 1A). Compared with WT-hiPSC-CMs, the fluorescent intensity and distribution of CDH2 were relatively much higher in the case of spontaneously and rhythmically beating CDH2-hiPSC-CMs.

Characterization of WT-hiPSC-CMs and CDH2-hiPSC-CMs. Immunostaining and fluorescence microscopic analysis of Day 28 WT-hiPSC-CM and CDH2-hiPSC-CM cultures revealed the expression pattern of human cardiac specific regulatory protein, troponin T (green, hcTnT) and cell adhesion protein, cadherin 2 (red, CDH2). Compared with WT-hiPSC-CMs, the fluorescent intensity and distribution of CDH2 were much higher in the spontaneously and rhythmically beating CDH2-hiPSC-CMs (A). Immunostaining and flowcytometric analysis of Day 28 WT-hiPSC-CM and CDH2-hiPSC-CM cultures demonstrated the expression profile of hcTnT (98.8% and 99.2%, respectively), indicating a high level of purity of the differentiated myocytes (B). Similarly, western blot analyses validated the protein expression profiles of CDH2 and a gap junction protein, connexin 43 (Cx43), in both types of cardiac myocytes (C). Relative quantification of proteins showed that the expression levels of CDH2 and Cx43 were significantly higher in the case of CDH2-hiPSC-CMs compared with WT-hiPSC-CMs (D). The housekeeping protein, GAPDH, was used for western blot normalization. Cells were also counterstained for nuclei (blue, DAPI). Scale bar = 20 µm (panels in A). The values were means ± SEM for five independent cultures (n = 4). (*WT-hiPSC-CMs < CDH2-hiPSC-CMs, P < 0.05), by Student’s t-test with Holm–Sidak’s multiple comparisons test (D).

As well as quantitative analysis of the same set of markers by flow cytometry, exhibited a high expression of hcTnT (WT-hiPSC-CMs, 98.8%; CDH2-hiPSC-Cms, 99.2%), which is a consistent characteristic of differentiating CMs (Figure 1 B). Likewise, western blot analyses validated the protein expression profiles of CDH2 and a gap junction protein (Cx43), in both categories of CMs (Figure 1 C). In addition, relative quantification of these proteins showed that the expression levels of CDH2 and Cx43 were significantly up-regulated in the case of CDH2-hiPSC-CMs compared with WT-hiPSC-CMs (Figure 1 D). These results indicated that the hiPSC-derived CMs viz., WT-hiPSC-CMs and CDH2-hiPSC-CMs, contained almost pure population of CMs, and the cultures were devoid of any other undifferentiated stem and/or progenitors.

3.2 Action potential propagation in WT-hiPSC-CMs and CDH2-hiPSC-CMs cell monolayers

In order to compare the AP propagation between WT-hiPSC-CMs and CDH2-hiPSC-CMs in confluent monolayer cultures, the optical mapping of APs were carried out (Figure 2 A and B). The activation spread was uniform and the shape of generated APs was similar, in both types of CMs. However, the measured conduction velocity was significantly higher (on an average by ∼48%) in CDH2-hiPSC-CM monolayers than in WT-hiPSC-CMs monolayers (17.3 ± 3.7 vs. 11.7 ± 2.3 cm/s, respectively, P < 0.005) (Figure 2 C). No statistically significant differences were observed between these two types of CMs with respect to the other measured parameters, viz., AP upstroke rise time (Figure 2 D), as well as AP duration (APD) at 50% (APD50) (Figure 2 E) and APD at 80% repolarization (APD80) (Figure 2 F), demonstrating that the cell excitable properties were similar in both types of CM cultures. Therefore, the observed increase of conduction velocity in CDH2-hiPSC-CMs can be attributed to an increase in the intercellular coupling.

Action potential propagation in WT-hiPSC-CMs and CHD2-hiPSC-CMs cell monolayers. Representative examples of action potentials (APs) propagation during pacing at a cycle length (CL) of 800 ms in WT-hiPSC-CMs (A) and CDH2-hiPSC-CMs (B), respectively. Phase-contrast microphotographs of typical Day 28 cultures, displaying the homogeneous confluence (A and B), ischronal maps of activation spread, conduction velocity (CV), activation time (AT), and selected traces of raw optical APs and a spatially averaged AP over a 7 × 7 diodes in the monolayer centre without the motion artefact (A and B). Red dots depict ATs, green and brown dots depict times of repolarization to 50% and 80% levels of amplitude (A and B). Conduction velocity was significantly higher (∼48%) in CDH2-hiPSC-CMs than in WT-hiPSC-CMs (17.3 ± 3.7 vs. 11.7 ± 2.3 cm/s) (C). No significant difference was observed between these two types of cardiac myocytes with respect to the other measured parameters, viz., action potential (AP) upstroke rise time (RT) (D), as well as AP duration (APD) at 50% repolarization (APD50) (E) and APD at 80% repolarization (APD80) (F). Scale bar = 100 µm (phase contrast images, panels in A, B). The values were means ± SEM for eight independent cultures (n = 8). (*WT-hiPSC-CMs < CDH2-hiPSC-CMs, P < 0.005), by Student’s t-test with Holm–Sidak’s multiple comparisons test (C–F).

3.3 CDH2 overexpression enhances survival and engraftment potential of hiPSC-CMs in vivo

To determine whether the effect of CDH2 overexpression could promote the survival and engraftment potential of hiPSC-CMs in vivo, and because the MI induced mice were implanted with myocytes (WT-hiPSC-CMs or CDH2-hiPSC-CMs) that constitutively expressed luciferase enzyme, and were of human origin, both bioluminescence imaging (BLI) and immunohistochemical (IHC) detection methods were employed (Figure 3 A and B). In post-MI, Day 7 and Day 28 animals, assessment of luciferin signal and percentage of survival cells were significantly greater in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group (P < 0.05) and were significantly higher in both cell-treatment groups than in untreated control MI animals (P < 0.05) (Figure 3 C and D).

Survival of implanted WT-hiPSC-CMs and CDH2-hiPSC-CMs *in vivo*. The MI induced mice were implanted with myocytes (WT-hiPSC-CMs or CDH2-hiPSC-CMs) that constitutively expressed luciferase enzyme. Known quantities of luciferase expressing myocytes, viz., WT-hiPSC-CMs or CDH2-hiPSC-CMs were imaged, and the collected data were utilized to generate a standard curve depicting the relationship between bioluminescent imaging intensity (BLI) and cell number (A). After 7 or 28 days of induction of MI, all animals received D-luciferin injections. Bioluminescence images were captured 10 min post D-luciferin injection (B). To determine the number of engrafted cells among various MI treated groups, the BLI signal intensity was compared with the generated standard curve (C). The engraftment rate/survival was calculated by dividing the number of cells determined via BLI by the total number of cells administrated and expressed as a percentage (D). The values were means ± SEM. The number of animals per group was 7–9 (n = 7–9). (*WT-hiPSC-CMs > MI, CDH2-hiPSC-CMs > MI, P < 0.05), (^#WT-hiPSC-CMs < CDH2-hiPSC-CMs, P < 0.05), by Student’s t-test with Holm–Sidak’s multiple comparisons test (C and D).

Similarly, in post-MI, Day 7 and Day 28 left ventricular sections, localization of key human cardiac myocyte and nuclear phenotypic markers revealed the expression pattern of hcTnT and human-specific nucleolar phosphoprotein, NCL. Immunostaining revealed varying degrees of survival and functional integration of implanted cells, viz., WT-hiPSC-CMs and CDH2-hiPSC-CMs, on Day 7 and Day 28 post-MI (Figure 4 A and B). The observed engraftment rate of CDH2-hiPSC-CMs was significantly higher than WT-hiPSC-CMs (P < 0.05) (Figure 4 C). As well as quantification of the percentage of CMs that were double positive for hcTnT and NCL in the engraftments, revealed no significant difference between these two-implanted cell groups, viz., WT-hiPSC-CMs and CDH2-hiPSC-CMs (Figure 4 D). These results demonstrated that in both cell treatment groups, over 93% of the engrafted cells were hiPSC-CMs (Figure 4 D).

Engraftment potential of WT-hiPSC-CMs and CDH2-hiPSC-CMs *in vivo*. In post-MI, Day 7 and Day 28 left ventricular sections, localization of key human cardiac myocyte and nuclear phenotypic makers illustrated the expression pattern of human cardiac specific regulatory protein, troponin T (red, hcTnT), and human-specific nucleolar phosphoprotein, nucleolin (fuchsia, NCL). Immunostaining revealed varying degrees of survival and functional integration of implanted cells, viz., WT-hiPSC-CMs and CDH2-hiPSC-CMs, on Day 7 (A) and Day 28 (B) post-MI. The engraftment rate of CDH2-hiPSC-CMs was significantly higher than WT-hiPSC-CMs (C). Quantification of the percentage of CMs that were double positive for hcTnT and NCL in the engraftments, revealed no statistically significant difference between the two-implanted groups, viz., WT-hiPSC-CMs and CDH2-hiPSC-CMs (D). Cells were also counterstained for nuclei (blue, DAPI). Scale bar = 20 µm (panels in A, B). The values were means ± SEM. The number of animals per group was 5–9 (n = 5–9). (*WT-hiPSC-CMs < CDH2-hiPSC-CMs, P < 0.05), by two-way ANOVA (C); and Student’s t-test with Holm–Sidak’s multiple comparisons test (D).

We have injected almost pure populations of luciferase expressing CMs based on the metabolic purification, still, there exist a possibility of the presence of luciferase expressing other differentiating/contaminating cells (i.e. non-CMs) that could confound the obtained results from our BLI measurements. Thus, we carefully determined the proportion of CMs and non-CMs based on the expression of both human nucleolin (NCL) and cardiac troponin T (cTnT), dual positivity. Our results confirmed that both WT-hiPSC-CMs and CDh2-hiPSC-CMs treatments revealed >93% of engrafted hiPSC-derived cells were, in fact, CMs (Figure 4 D).

3.4 Detection and histological evaluation of hiPSC-CMs in engraftment and remote zone

Next, localization of key cardiomyogenic markers viz., CDH2, cTnT, Cx43, and NCL, in post-MI Day 7 (Supplementary material online, Figures S2 and S4) and Day 28 (Supplementary material online, Figures S3 and S5) ventricular sections was performed. Human-specific nucleolin, NCL, was solely detected in engraftment zones (Supplementary material online, Figures S2A–S5A), i.e. the infarct and border zones but not in the remote zone (Supplementary material online, Figures S2B–S5B), i.e. the viable myocardium of left ventricular sections. The expression levels of both CDH2 and Cx43 were relatively up-regulated in CDH2-hiPSC-CMs engraftment group compared with WT-hiPSC engraftment group, both in Day 7 and in Day 28 tissue sections, signifying that CDH2 overexpression enabled the formation of a well-coupled electro-mechanical syncytium CMs.

3.5 Induction and assessment of neo-vasculogenic response around peri-infarct border zone

The peri-infarct border zones were evaluated for hiPSC-CMs induced neo-angiogenic response by dual immunostaining for cardiac and endothelial phenotypic markers, viz., cTnT and isolectin B4, in post-MI, Day 7 (Supplementary material online, Figure S6) and Day 28 (Figure 5) ventricular sections. The vessel density was quantified as the number of isolectin B4-positive vascular structures per square millimetre (Supplementary material online, Figure S6A; Figure 5 A). The untreated MI group (MI) and the treated MI groups (WT-hiPSC-CMs and CDH2-hiPSC-CMs) showed significantly lower vascular density (VD) compared with the sham-operated animals. Besides, the neo-angiogenic response was significantly higher in both cell treatment groups than in untreated control MI animals. However, the quantified VD was significantly less in the WT-hiPSC-CM treated MI group than in CDH2-hiPSC-CM treated MI group (Supplementary material online, Figure S6B; Figure 5 B).

Evaluation of peri-infarct border zone neo-vasculogenic response. In post-MI, Day 28 left ventricular peri-infarct border zone sections, immunolocalization of cardiac and endothelial specific phenotypic markers exhibited the expression pattern of cardiac specific regulatory protein, troponin T (green, cTnT), and lectin, (red, isolectin B4) (A). The per-infarct border zone neo-vasculogenic response was evaluated; the vessel density was quantified as the number of isolectin B4-positive vascular structures per square millimetre (B). The untreated MI group (MI) and treated MI groups (WT-hiPSC-CMs and CDH2-hiPSC-CMs) showed significantly higher vascular density (VD) compared with sham operated animals. Besides, the neo-angiogenic response was significantly higher in both cell-treatment groups than in untreated control MI animals. However, the quantified VD was significantly less in the WT-hiPSC-CM treated MI group than in CDH2-hiPSC-CM treated MI group (B). Cells were also counterstained for nuclei (blue, DAPI). Scale bar = 20 µm (panels in A). The values were means ± SEM. The number of animals per group was 5–7 (n = 5–7). (^†Sham < MI, WT-hiPSC-CMs, and CDH2-hiPSC-CMs, P < 0.05), (*MI < WT-hiPSC-CMs, and CDH2-hiPSC-CMs, P < 0.05), (^#CDH2-hiPSC-CMs > WT-hiPSC-CMs), by one-way ANOVA, non-parametric Kruskal–Wallis test (B).

In recent years, it has been shown that CDH2 could promote the expression of vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen, which activates the ERK 1/2 signalling pathway.⁴¹ Protein analysis by western blot exhibited the expression pattern of VEGF and activation of ERK (extracellular signal-regulated kinases) signalling pathway, in both WT-hiPSC-CMs and CDH2-hiPSC-CMs (Supplementary material online, Figure S7A). Semi-quantitative Western blot analysis demonstrated that the levels of ERK activation, i.e. the expression levels of phosphorylated ERK (pERK), were significantly greater in CDH2-hiPSC-CMs compared with WT-hiPSC-CMs (Supplementary material online, Figure S7B). Likewise, the expression of VEGF molecule was significantly up-regulated in CDH2-hiPSC-CMs than in WT-hiPSC-CMs (Supplementary material online, Figure S7C).

3.6 CDH2 overexpression promotes the therapeutic efficacy of hiPSC-CMs in vivo

To establish whether the effect of CDH2 overexpression in cultured hiPSC-CMs could increase the CMs’ therapeutic efficacy during myocardial recovery, using a MI model once again, the cardioprotective potential of WT-hiPSC-CMs and CDH2-hiPSC-CMs were evaluated, at post-MI Day 7 and Day 28. Using Sirus Red/Fast Green histochemical staining, the areas of infarcted (red, non-viable) and non-infarcted (green, viable) zones, were identified, in post-MI Day 7 and Day 28 ventricular tissue sections (Figure 6A). Subsequently, the infarct size was quantified as the ratio of the scar area to the total surface area of the left ventricle and expressed as a percentage, for Day 7 (Figure 6 B) and for Day 28 (Figure 6 C). At Day 7, the CDH2-hiPSC-CMs treatment group showed significant reduction in infract size compared with the either the WT-hiPSC-CMs treatment group or the untreated control MI animals (Figure 6 B). While, at Day 28, the infarct size was significantly reduced in the CDH2-hiPSC-CMs treated MI group than in the WT-hiPSC-CMs treated MI group, and in both cell treatment groups than in untreated control MI animals (Figure 6 C). Echocardiographic assessments of left ventricular function, such as ejection fraction (Figure 6 D) and fractional shortening (Figure 6 E), were performed before MI induction (pre-MI) and on post-MI Day 7 and Day 28. Ejection fraction/fractional shortening were significantly greater in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group and were significantly less in both cell-treatment groups than in pre-MI induction of the same animals. Taken together, these results would indicate that CDH2 overexpression, in fact, may promote the therapeutic efficacy of hiPSC-CMs during myocardial reparative process.

Assessment of left ventricular function by echocardiography. Sirus Red/Fast Green histochemical staining, revealing areas of infarcted (red, non-viable) and non-infarcted (green, viable) zones, in post-MI Day 7 and Day 28 ventricular tissue sections (A). The infarct size was quantified as the ratio of the scar area to the total surface area of the left ventricle and expressed as a percentage, for Day 7 (B) and for Day 28 (C). At Day 7, the CDH2-hiPSC-CMs treatment group showed significant reduction in infract size compared with the either the WT-hiPSC-CMs treatment group or the untreated control MI animals (B). While at Day 28, the infarct size was significantly reduced in the CDH2-hiPSC-CMs treated MI group than in the WT-hiPSC-CMs treated MI group, and in both cell-treatment groups than in untreated control MI animals. Echocardiographic assessments of left ventricular function, such as ejection fraction (D) and fractional shortening (E), were performed before MI induction (pre-MI) and on post-MI Day 7 and Day 28. Ejection fraction/fractional shortening were significantly greater in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group, and were significantly less in both cell-treatment groups than in pre-MI induction of the same animals. Scale bar = 1000 µm (panels in A). The values were means ± SEM. The number of animals per group was 7–9 (n = 7–9). (Panel B: *MI and WT-hiPSC-CMs > CDH2-hiPSC-CMs, P < 0.05; ^#WT-hiPSC-CMs > CDH2-hiPSC-CMs, P < 0.05), (Panel C: *MI > WT-hiPSC-CMs and CDH2-hiPSC-CMs, P < 0.05; ^#WT-hiPSC-CMs > CDH2-hiPSC-CMs, P < 0.05). (Panels D and E: *MI > WT-hiPSC-CMs and CDH2-hiPSC-CMs, P < 0.05; ^#WT-hiPSC-CMs > CDH2-hiPSC-CMs, P < 0.05), by one-way ANOVA, non-parametric Kruskal–Wallis test (B, C), and by two-way ANOVA (D, E).

3.7 Evaluation of WT-hiPSC-CMs and CDH2-hiPSC-CMs apoptotic response under hypoxic conditions in vitro

To understand the mechanism by which overexpression of CDH2 promotes the survival and engraftment of implanted myocytes, i.e. the WT-hiPSC-CMs and CDH2-hiPSC-CMs; the cells were subjected to hypoxic conditions over a period of 48 h. TUNEL staining followed by immunostaining of hcTnT, revealed the nuclear positivity of apoptotic CMs along with the cytoplasmic positivity of hcTnT, after 48 h of hypoxic treatment (Figure 7 A). CDH2-hiPSC-CMs showed significantly fewer number of TUNEL positive cells than WT-hiPSC-CMs, at 12, 24, or 48 h of hypoxic treatment (P < 0.005) (Figure 7 B). Protein expression analysis by western blot revealed the activation pattern of AKT (a serine/threonine kinase or protein kinase B) signalling pathway as a function of time, in both WT-hiPSC-CMs and CDH2-hiPSC-CMs (Figure 7 C). The levels of AKT activation were significantly higher in CDH2-hiPSC-CMs than in WT-hiPSC-CMs (P < 0.005) (Figure 7 D). The normoxic treated myocytes served as controls. These results would signify that CDH2 overexpression enhances the anti-apoptotic tendencies of the hiPSC-CMs under hypoxic conditions by activating AKT signalling pathway.

Detection and quantification of apoptosis by TUNEL assay. Detection and quantitative analysis of myocytes (WT-hiPSC-CMs or CDH2-hiPSC-CMs) treated under hypoxic conditions over a period of 48 h and measured by TUNEL [terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling] assay. TUNEL staining, revealing the nuclear positivity of apoptotic cardiac myocytes (red, TUNEL) and the human cardiac specific regulatory protein, troponin T (green, hcTnT), after 48 h of hypoxic treatment (A). CDH2-hiPSC-CMs showed significantly less number of TUNEL positive cells than WT-hiPSC-CMs, at 12, 24, or 48 h of hypoxic treatment (B). Semi-quantitative western blot analysis, illustrating the activation pattern of AKT signalling pathway as a function of time, in both WT-hiPSC-CMs and CDH2-hiPSC-CMs (C). The levels of AKT activation were significantly higher in CDH2-hiPSC-CMs than in WT-hiPSC-CMs (D). The normoxic treated myocytes served as controls. (E) Illustration of activation of AKT-/ERK-/connexin-dependent signal transduction pathways by CDH2, results in enhancement of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts. Cells were also counterstained for nuclei (blue, DAPI). Scale bar = 20 µm (Panels in A). The values were means ± SEM for three to five independent cultures (D, n = 3; B, n = 5). (Panel B: *WT-hiPSC-CMs > CDH2-hiPSC-CMs, P < 0.005), (Panel C: *WT-hiPSC-CMs < CDH2-hiPSC-CMs, P < 0.005), by two-way ANOVA (B, D).

In conclusion, these data suggest that activation of AKT-/ERK-/connexin-dependent signal transduction pathways by CDH2, results in enhancement of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts (Figure 7 E).

3.8 mRNA analysis of WT-hiPSC-CMs and CDH2-hiPSC-CMs: an in vitro assessment of pluripotency and cardiomyogenesis

To compare the differential gene expression profile between WT-hiPSC-CMs culture vs. CDH2-hiPSC-CMs culture, RT-qPCR analysis of significant pluripotent and early stage cardiomyogenic differentiation markers was carried out at various defined time points (Supplementary material online, Figure S8A–H). The pluripotent transcription factors, viz., Oct3/4, Sox2, and Nanog, showed initial up-regulation around Day 0 followed by progressive down-regulation around Day 5 of differentiation (D-Day 5), and were almost undetectable by Day 7 (B-Day 7) and day 30 (B-Day 30), i.e. after the spontaneous beating was first observed in these cultures (Supplementary material online, Figure S8A–C). On the contrary, the cardiomyogenic transcription factors, viz., brachyury, Mef2C, and Mesp1, showed peak levels of expression between Day 5 of differentiation (D-Day 5) and the first day of beating (B-Day 1). Similarly, the cardiac-specific transcription factors, viz., Gata4 and Tbx5, showed sustained up-regulation until Day 7 (B-Day 7), with a noticeable kinetics of gradual down-regulation of these transcripts over the consecutive experimental time point, i.e. around Day 30 (B-Day 30) (Supplementary material online, Figure S8D–H).

We have utilized a purified population of CDH2 overexpressing CMs, and since these differentiated CMs were generated by means of lentiviral transduction, we would like to confirm whether the CDH2 overexpressing cells did not express the pluripotency markers due to off-target effects of transfection. In addition, the differentiated CMs should not revert to a primitive cellular status (dedifferentiation or partial dedifferentiation) and/or turning into malignant cells. Thus, the results were indicative of the absence of any undifferentiated stem and/or progenitors but also suggestive of the modulation of cardiomyogenic gene expression in this milieu, and consequently, driving myocardial specification, differentiation, and maturation.

4. Discussion

Stem cell-based cardiac repair is one of the promising therapeutic modality for inducing the repair and/or regeneration of damaged or necrotic myocardium.⁴² Nevertheless, so far, experimental studies addressing the capacity of transplanted stem and/or progenitors to engraft and differentiate into cardiomyogenic lineage yielded conflicting results.⁴ ^, ⁶ In this study, we report that CDH2 overexpression can augment the regenerative and/or reparative properties of hiPSC-derived cardiac progenitors, viz., the CDH2-hiPSC-CMs. We have demonstrated unequivocally that the genetic enhancement of hiPSC-CMs improved not only the survival/engraftment of cultured myocytes but also the electrical connectivity and functional integration of these cells, consequently, an improvement of the reparative properties of implanted CDH2-hiPSC-CMs in the failing heart.

Here, we have shown that several molecular mechanisms are involved in the functional recovery of MI animals when implanted with CDH2-hiPSC-CMs. Compared with WT-hiPSC-CMs, firstly, the CDH2-hiPSC-CMs are able to survive better not only under hypoxic conditions in vitro but also under myocardial ischaemic conditions in vivo, especially through the activation of AKT signalling pathway. Second, the CDH2-hiPSC-CMs are able to induce robust neo-angiogenesis around peri-infarct areas by means of paracrine mechanisms, specifically through VEGF-mediated activation of ERK signalling pathway. Finally, the CDH2-hiPSC-CMs are better able to integrate in the damaged myocardium, particularly due to the CDH2-mediated up-regulation of the transmembrane channels (connexons), developing into an electromechanical syncytium. Thus, these various intrinsic attributes govern the enhanced reparative properties of CDH2-hiPSC-CMs.

The AKT pathway is an intracellular signal transduction pathway that has pleiotropic effects on cell survival, growth, proliferation, metabolism, and angiogenesis in response to extracellular signals. AKT is expressed in various types of cells, including cancer cells, neuronal cells, and stem cells. Cadherin 2 (CHD2) has been reported to exert an anti-apoptotic effect, by activating AKT signalling pathway.^43–45 Because PI3K/AKT signalling pathway is another downstream of CDH2, it has been shown, that overexpression of CDH2 can result in activation of PI3K/AKT/GSK3b pathway; leading to elevated expression of AKT that eventually promotes the survival of cells.⁴⁶ ^, ⁴⁷ AKT inhibits transcription factors that promote the expression of pro-apoptotic genes and enhances transcription of anti-apoptotic genes. In addition, reduction of CDH2 expression can lead to a decrease in phosphorylation of AKT.⁴⁴ In this study, we have provided compelling evidence that overexpression of CHD2 could significantly up-regulate the expression of pAKT, resulting in decreased apoptotic cellular death of hiPSC-CMs under hypoxic conditions over a period of 48 h, in vitro. Thus, the strategy of enhancing the expression of CDH2 in hiPSC-CMs under hypoxic condition may naturally facilitate the survival rate of these cells.

In our culture conditions, we did not observe any decrease in the levels of expression of CDH2 (N-cadherin) over the course of differentiation, i.e. on Day 7 and Day 28, not only in the case of α-MHC-driven CDH2-hiPSC-CMs but also in the case of WT-hiPSC-CMs. Consequently, no enhanced apoptotic tendency of these cells was evident in our experimental conditions, both in vitro as well as in vivo. Thus, as the CMs mature, the α-MCH-driven CDH2 expression was not down-regulated as the cells undergo progressive maturation; this could possibly due to a narrow window of differentiation time under study. However, using hESC-derived CMs, Kita-Matsuo et al.⁴⁸ have shown that the onset of endogenous and transgene expression of αMHC could be evident as CMs emerge and persist for at least 120 days. Conversely, the initial detection of the bMHC transcripts in these hESC-derived CMs was at Day 90.⁴⁸

A number of studies have shown that paracrine release of VEGF by grafted and/or implanted CMs, promoted the angiogenesis.³⁴ ^, ³⁷ ^, ⁴⁹ CDH2 has been reported to promote the expression of VEGF through ERK activation. ERK-VEGF axis is a downstream of CDH2. It has been that CDH2-mediated cell–cell contact (overexpression) promotes the expression of VEGF through ERK activation, especially in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs).⁴¹ Furthermore, CDH2 blocking antibody could suppress the ERK activity leading to the down-regulation of VEGF.⁴¹ Taken together, these results suggest that CDH2 promotes ERK activation that in turn increased the expression of VEGF in hUCB-MSCs. So, by evaluating the protein expression of ERK, pERK, and VEGF, we found that CDH2-hiPSC-CMs showed a significant up-regulation of VEGF through activation of ERK pathway. This is corroborated also by the histological analysis of isolectin-B4-stained left ventricular infarct tissue sections, the CDH2-hiPSC-CMs treatment group showed a robust induction of neo-vasculogenesis around the peri-infarct zone over a period of 28 days. Thus, the relatively modest improvement of left ventricular function, post-MI, may be attributed to mere paracrine effects of implanted cells, resulting in the release of proangiogenic and anti-apoptotic factors.

CDH2 is required not only for the formation and/or function of gap junctions but also for the stability of gap junction proteins, such as connexin 43.²⁴ ^, ²⁶ ^, ²⁷ ^, ⁵⁰ In addition, CDH2 knockout cardiomyocytes result in, disassembly of gap junctions, increased dephosphorylation of connexin 43 (inactive form), and decreased connexin 43 expression.²⁴ Collectively, these facts suggest that CDH2-mediated up-regulation of the transmembrane channels (connexons), may facilitate formation of a functional electromechanical syncytium.

Electro-mechanical integration of implanted CMs are essential, not only to enable a functional integration of implanted and native CMs but also to enhance the efficacy of cardiac cell therapy.⁴⁹ ^, ⁵¹ ^, ⁵² In order to achieve this result, the grafted CMs are required to express the cell junction proteins, viz., CDH2 (adhesion protein) and Cx43 (gap junction), along the intercalated disks. These intercellular connections are crucial for electrical propagation and bi-directional force transmission between the grafted cells and the host-myocardium.⁵³ It has been shown that, on the one hand, suboptimal level of Cx43 at the host-iPSC-CM interface, can lead to insufficient electrical coupling of graft and native CMs that may incur the risk of proarrhythmias⁵¹ ^, ⁵⁴; but on the other hand, overexpression of Cx43 can considerably increase the conduction velocity of CMs. Since Cx43 expression is tightly linked to CDH2 expression, in our study, we have provided compelling evidence that CDH2 overexpression in hiPSC-CMs significantly enhanced conduction velocity of CMs in vitro. Thus, we demonstrated that the genetic enhancement of hiPSC-CMs, i.e. overexpression of CDH2 in hiPSC-CMs, may be an effective biological strategy for enhancing homotypic cell–cell electrical coupling (grafted hiPSC-CMs and host-CMs), and may lower the chance of developing arrhythmias in vivo.

Finally, the vital issue for realistic clinical application is whether these genetically modified CMs, CDH2-hiPSC-CMs, can undergo maturation into terminally differentiated adult CMs. Several strategies have been employed to improve the maturation and differentiation of hiPSC-CMs, including long-term culture, electrical and/or mechanical stimulation.²⁰ ^, ^55–57 Recently, Ronaldson-Bouchard et al.⁵⁸ have demonstrated that adult-like myocardial tissue can be generated from hiPSC-CMs in fibrin hydrogel subjected to physical conditioning, i.e. stretch and auxotonic contractions, within a span of just four weeks of in vitro culture. Further experiments are warranted to promote maturity of CDH2-hiPSC-CMs/WT-hiPSC-CMs; thus, to address this critical issue, in vitro experiments are underway to ascertain whether mechanical and/or electrical stimulation can propel CDH2-hiPSC-CMs maturation, using a combination of sex-mismatched and/or genetically or fluorescently marked cells.

In summary, here, we report the generation of genetically enhanced MHC-promoter driven hiPSC-CMs, i.e. CDH2-hiPSC-CMs, consisting of all the essential characteristics of a cellular cardiac graft, such as spontaneous and synchronized contractility, stable electrophysiological properties, ability to induce neo-vasculogenesis, to address the most important problem besetting the cardiac therapy until now, i.e. the repair and/or regeneration of injured myocardium.

This study is innovative on several fronts; first, it uses CDH2-hiPSC-CMs for enhanced homotypic cell–cell coupling, i.e. mechanical and electrical coupling between engrafted and host-CMs. Second, activation of paracrine mechanisms by CDH2-hiPSC-CMs, such as induction of neo-vasculogenesis and expression of anti-apoptotic genes, resulting in a higher survival/engraftment rate of implanted cells. Probably, this increase in paracrine activity may possibly contribute to the improvement in left ventricular function and remodelling, which ultimately, resulted in a remarkable angiogenesis at peri-infarct border zones, culminating into a sizable reduction of infract size.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

Funding

This work was supported by the National Institute of Health (NHLBI R01 grants: HL95077, HL114120, HL131017, and UO1 HL 134764) for J.Z.

Supplementary Material

cvz179_Supplementary_Material

Click here for additional data file.^{(5MB, pdf)}

Time for primary review: 21 days

Translational perspective

Our study proposes to combine cellular cardiomyoplasty with gene therapy to improve left ventricular function and remodelling after myocardial infarction (MI). The reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes could be enhanced through CDH2 overexpression. This unique approach displays several features required for clinical application, which would open new perspectives in clinical setting for the effective management of MI, and thus, can lead to functional enhancement of a failing heart.

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Supplementary Materials

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[cvz179-B58] 58. Ronaldson-Bouchard K, Ma SP, Yeager K, Chen T, Song L, Sirabella D, Morikawa K, Teles D, Yazawa M, Vunjak-Novakovic G. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 2018;556:239–243. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

N-cadherin overexpression enhances the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes in infarcted mouse hearts

Xi Lou

Meng Zhao

Chengming Fan

Vladimir G Fast

Mani T Valarmathi

Wuqiang Zhu

Jianyi Zhang

Abstract

Aims

Methods and results

Conclusion

Graphical Abstract

Graphical Abstract.

1. Introduction

2. Methods

2.1 WT-hiPSC culture: expansion and maintenance

2.2 CDH2-hiPSCs culture: generation, expansion, and maintenance

2.3 Cardiomyogenic differentiation: generation of WT-hiPSC-CMs and CDH2-hiPSC-CMs

2.4 Purification and metabolic selection of WT-hiPSC-CMs and CDH2-hiPSC-CMs

2.5 Phenotypic characterization of WT-hiPSC-CMs and CDH2-hiPSC-CMs by single-colour flow cytometry

2.6 Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)

2.7 Optical mapping of action potentials (APs)

2.8 Mouse myocardial infarction model: WT-hiPSC-CMs and CDH2-hiPSC-CMs implantation

2.9 Echocardiography

2.10 Determination of infarct size

2.11 Immunostaining and fluorescence microscopy

2.12 Engraftment rate of WT-hiPSC-CMs and CDH2-hiPSC-CMs

2.13 Hypoxic treatment of WT-hiPSC-CMs and CDH2-hiPSC-CMs

2.14 Detection of apoptotic cells by TUNEL assay

2.15 Quantification of angiogenesis

2.16 Western blotting

2.17 Statistical analysis

3. Results

3.1 Phenotypic characterization of WT-hiPSC-CMs and CDH2-hiPSC-CMs

Figure 1.

3.2 Action potential propagation in WT-hiPSC-CMs and CDH2-hiPSC-CMs cell monolayers

Figure 2.

3.3 CDH2 overexpression enhances survival and engraftment potential of hiPSC-CMs in vivo

Figure 3.

Figure 4.

3.4 Detection and histological evaluation of hiPSC-CMs in engraftment and remote zone

3.5 Induction and assessment of neo-vasculogenic response around peri-infarct border zone

Figure 5.

3.6 CDH2 overexpression promotes the therapeutic efficacy of hiPSC-CMs in vivo

Figure 6.

3.7 Evaluation of WT-hiPSC-CMs and CDH2-hiPSC-CMs apoptotic response under hypoxic conditions in vitro

Figure 7.

3.8 mRNA analysis of WT-hiPSC-CMs and CDH2-hiPSC-CMs: an in vitro assessment of pluripotency and cardiomyogenesis

4. Discussion

Supplementary material

Funding

Supplementary Material

Translational perspective

References

Associated Data

Supplementary Materials

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