Dataset Information

Activation of MG53 Enhances Cell Survival and Engraftment of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Injured Hearts.

ABSTRACT:

Background and objective

Our previous studies demonstrated that MG53 protein can protect the myocardium, but its use as a therapeutic is challenging due to its short half-life in blood circulation. This study aimed to investigate the cardioprotective role of MG53 on human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) in the context of myocardial ischemia/reperfusion (I/R).

Methods

In vitro: HiPSC-CMs were transfected with adenoviral MG53 (HiPSC-CMs^MG53), in which the expression of MG53 can be controlled by doxycycline (Dox), and the cells were then exposed to H₂O₂ to mimic ischemia/reperfusion injury. In vivo: HiPSC-CMs^MG53 were transplanted into the peri-infarct region in NSG™ mice after I/R. After surgery, mice were treated with Dox (+ Dox) to activate MG53 expression (sucrose as a control of -Dox) and then assessed by echocardiography and immunohistochemistry.

Results

MG53 can be expressed in HiPSC-CM^MG53 and released into the culture medium after adding Dox. The cell survival rate of HiPSC-CM^MG53 was improved by Dox under the H₂O₂ condition. After 14 and 28 days of ischemia/reperfusion (I/R), transplanted HiPSC-CMs^MG53 + Dox significantly improved heart function, including ejection fraction (EF) and fractional shortening (FS) in mice, compared to HiPSC-CMs^MG53-Dox, and reduced the size of the infarction. Additionally, HiPSC-CM^MG53 + Dox mice demonstrated significant engraftment in the myocardium as shown by staining human nuclei-positive cells. In addition, the cell survival-related AKT signaling was found to be more active in HiPSC-CM^MG53 + Dox transplanted mice's myocardium compared to the HiPSC-CM^MG53-Dox group. Notably, the Dox treatment did not cause harm to other organs.

Conclusion

Inducible MG53 expression is a promising approach to enhance cell survival and engraftment of HiPSC-CMs for cardiac repair.

SUBMITTER: Park KH

PROVIDER: S-EPMC10579131 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Publications

Activation of MG53 Enhances Cell Survival and Engraftment of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Injured Hearts.

Park Ki Ho KH He Xingyu X Jiang Lin L Zhu Hua H Liang Jialiang J Wang Yigang Y Ma Jianjie J

Stem cell reviews and reports 20230721 7

<h4>Background and objective</h4>Our previous studies demonstrated that MG53 protein can protect the myocardium, but its use as a therapeutic is challenging due to its short half-life in blood circulation. This study aimed to investigate the cardioprotective role of MG53 on human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) in the context of myocardial ischemia/reperfusion (I/R).<h4>Methods</h4>In vitro: HiPSC-CMs were transfected with adenoviral MG53 (HiPSC-CMs<sup>MG53</sup ...[more]

PMID: 37477774

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Project description:Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.

Project description:BackgroundAlthough human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.MethodsiPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).ResultsWe showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.ConclusionsOverexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Dataset Information

Activation of MG53 Enhances Cell Survival and Engraftment of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Injured Hearts.

Background and objective

Methods

Results

Conclusion

Publications

Activation of MG53 Enhances Cell Survival and Engraftment of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Injured Hearts.

Similar Datasets

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