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Cell therapy with embryonic stem cell-derived cardiomyocytes encapsulated in injectable nanomatrix gel enhances cell engraftment and promotes cardiac repair.

ABSTRACT: A significant barrier to the therapeutic use of stem cells is poor cell retention in vivo. Here, we evaluate the therapeutic potential and long-term engraftment of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated in an injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand Arg-Gly-Asp-Ser (PA-RGDS) in experimental myocardial infarction (MI). We cultured rat neonatal CMs in PA-RGDS for 7 days and found that more than 90% of the CMs survived. Next, we intramyocardially injected mouse CM cell line HL-1 CMs with or without PA-RGDS into uninjured hearts. Histologic examination and flow cytometry analysis of digested heart tissues showed approximately 3-fold higher engraftment in the mice that received CMs with PA-RGDS compared to those without PA-RGDS. We further investigated the therapeutic effects and long-term engraftment of mESC-CMs with PA-RGDS on MI in comparison with PBS control, CM-only, and PA-RGDS only. Echocardiography demonstrated that the CM-only and CM+PA-RGDS groups showed higher cardiac function at week 2 compared to other groups. However, from 3 weeks, higher cardiac function was maintained only in the CM+PA-RGDS group; this was sustained for 12 weeks. Confocal microscopic examination of the cardiac tissues harvested at 14 weeks demonstrated sustained engraftment and integration of mESC-CMs into host myocardium in the CM+PA-RGDS group only. This study for the first time demonstrated that PA-RGDS encapsulation can enhance survival of mESC-derived CMs and improve cardiac function post-MI. This nanomatrix gel-mediated stem cell therapy can be a promising option for treating MI.

SUBMITTER: Ban K

PROVIDER: S-EPMC4212793 | biostudies-other | 2014 Oct

REPOSITORIES: biostudies-other

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Project description:In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-β treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275 ± 36 ms and conduction velocity (CV) of 10.6 ± 4.2 cm/s (n = 3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4 ± 0.9 cm/s, n = 35 vs. 30.1 ± 3.2 cm/s, n = 20 in the longitudinal direction and 4.3 ± 0.3 cm/s vs. 9.3 ± 0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6 ± 1.3 cm/s and 3.1 ± 0.5 cm/s, n = 11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.

Project description:Cardiomyocytes differentiated from human embryonic stem cells (hESCs) represent a promising cell source for heart repair, disease modeling and drug testing. However, improving the differentiation efficiency and maturation of hESC-derived cardiomyocytes (hESC-CMs) is still a major concern. Retinoic acid (RA) signaling plays multiple roles in heart development. However, the effects of RA on cardiomyocyte differentiation efficiency and maturation are still unknown. Methods: RA was added at different time intervals to identify the best treatment windows for cardiomyocyte differentiation and maturation. The efficiency of cardiomyocyte differentiation was detected by quantitative real-time PCR and flow cytometry. Cardiomyocytes maturation was detected by immunofluorescence staining, metabolic assays and patch clamp to verify structural, metabolic and electrophysiological maturation, respectively. RNA sequencing was used for splicing analysis. Results: We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of TNNT2, NKX2.5 and MYH6 on day 10 of differentiation. In addition, flow cytometry showed that the proportion of differentiated cardiomyocytes in the RA-treated group was significantly higher than that in control group. RA treatment on days 15-20 increased cardiomyocyte area, sarcomere length, multinucleation and mitochondrial copy number. RNA sequencing revealed RA promoted RNA isoform switch to the maturation-related form. Meanwhile, RA promoted electrophysiological maturation and calcium handling of hESC-CMs. Importantly, RA-treated cardiomyocytes showed decreased glycolysis and enhanced mitochondrial oxidative phosphorylation, with the increased utilization of fatty acid and exogenous pyruvate but not glutamine. Conclusion: Our data indicated that RA treatment at an early time window (days 2-4) promotes the efficiency of cardiomyocyte differentiation and that RA treatment post beating (days 15-20) promotes cardiomyocyte maturation. The biphasic effects of RA provide new insights for improving cardiomyocyte differentiation and quality.

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Cell therapy with embryonic stem cell-derived cardiomyocytes encapsulated in injectable nanomatrix gel enhances cell engraftment and promotes cardiac repair.

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