Abstract Body:

Introduction

In stem cell-based therapy for cardiac regeneration, therapeutic cell populations are injected into the heart with the primary goal of reducing myocardial scar. Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) have shown great promise in promoting cardiac regeneration in animal models of myocardial infarction (1-4). However, obstacles remain in advancing PSC-CMs therapies, one of which is the low survival rate of transplanted cells. Numerous strategies have been investigated to improve cell retention, such as incorporation of biomaterials, genetic modification to regulate relevant genes, supplementation with cytokines or growth factors, and enhancement of in-vitro maturation (5). To turly enable cell therapy optimization and translation, a non-invasive cell tracking technology is needed to inform on cell survival, distribution, and integration in deep tissue. No current imaging solution for cell tracking is compatible with long-term monitoring in vivo. The objective of this study was to apply a novel bright-ferritin magnetic resonance imaging (MRI) mechanism to track PSC-CM transplants longitudinally and on-demand in the rat heart (6).

Methods

Human embryonic stem cells (hESCs) ESI-017 were genetically modified to stably overexpress ferritin using a CRISPR-Cas9 system (6). Wild-type and ferritin-overexpressing hESCs were differentiated into cardiomyocytes in vitro (7-8). The effect of ferritin overexpression and manganese (Mn) supplementation on hESC-derived cardiomyocytes (hESC-CMs) was evaluated based on cell viability and morphological and functional phenotypes. In-vitro MRI was performed to investigate the efficiency of the bright-ferritin mechanism. In vivo, 10 million cells were injected into the myocardium in the left ventricle of female Rowett athymic nude (RNU) rats. Longitudinal cell tracking using bright-ferritin was investigated out to 8 weeks post-cell transplantation, with Mn supplementation administered on-demand to recall bright signal. Cardiovascular MRI was acquired using retrospective gating on a 3 Tesla preclinical scanner (MR Solutions, Guildford, UK). Multi-slice CINE images in the short-axis view were reconstructed using a custom-built software (9). MRI findings will be corroborated against histological validation.

Results

Ferritin-overexpressing human embryonic stem cells (hESCs) were generated using a CRISPR/Cas9 system. Ferritin overexpression was preserved after differentiation into cardiomyocytes (Figure 1A-B). Ferritin-overexpressing hESC-derived cardiomyocytes (Ferritin hESC-CMs) displayed similar morphological phenotypes in terms of contractile markers as wild-type (WT) hESC-CMs (Figure 1C). MnCl₂ dosing tests revealed that both WT hESC-CMs and Ferritin hESC-CMs exhibited significant cell death when supplemented with 0.1 mM MnCl₂ for 24 h (Figure 1D). However, there was no adverse affect on cell viability when supplemented with 0.1 mM MnCl₂ for 2 h and 6 h (Figure 1D). With 2 h of 0.1 mM MnCl₂ supplementation, significant bright contrast from Ferritin hESC-CMs was obtained on in-vitro MRI (Figure 1E). Upon MnCl₂ supplementation, Ferritin hESC-CMs displayed a T1 relaxation time that was 1.5-times lower than that of WT hESC-CMs (Figure 1F). In vivo, rats who received intramyocardial transplantation of Ferritin hESC-CMs displayed bright contrast at the cell injections after Mn supplementation out to 8 weeks (Figure 1G). Histology confirmed the presence of viable transplanted cells.

Conclusions

Human embryonic stem cell-derived cardiomyocytes injected in the rat heart could be tracked longitudinally and on-demand via a bright T1-contrast on MRI.

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