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Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

ABSTRACT: The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca²⁺ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0?±?3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8?±?1.1 cm/s on days 1- 2 to 3.3?±?1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

SUBMITTER: Mattapally S

PROVIDER: S-EPMC6139622 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

Mattapally Saidulu S Zhu Wuqiang W Fast Vladimir G VG Gao Ling L Worley Chelsea C Kannappan Ramaswamy R Borovjagin Anton V AV Zhang Jianyi J

American journal of physiology. Heart and circulatory physiology 20180406 2

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were o ...[more]

PMID: 29631371

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Project description:BackgroundHuman induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) exhibit the potential to repair the injured heart after myocardial infarction (MI) by promoting neovascularization and cardiomyocyte survival. However, because of the low cellular retention and poor engraftment efficacy, cell therapy of MI is partly mediated by exosomes secreted from the transplanted cells. In this study, we investigated whether exosomes secreted from hiPSC-ECs could become a promising acellular approach to repair the infarcted heart after MI and elucidated the underlying protective mechanism.MethodsThe hiPSC-ECs were differentiated, and exosomes were isolated in vitro. Then, hiPSC-EC exosomes were delivered by intramyocardial injection in a murine MI model in vivo. Echocardiography, combined with hemodynamic measurement, histological examination, Ca2+ transient and cell shortening assessment, and Western blot, was used to determine the protective effects of hiPSC-EC exosomes on the infarcted heart. Furthermore, microRNA sequencing, luciferase activity assay, and microRNA gain-loss function experiments were performed to investigate the enriched microRNA and its role in exosome-mediated effects.ResultsIn vitro, the hiPSC-EC exosomes enhanced intracellular Ca2+ transients, increased ATP content, and improved cell survival to protect cardiomyocytes from oxygen-glucose deprivation injury. Congruously, hiPSC-EC exosome administration in vivo improved the myocardial contractile function and attenuated the harmful left ventricular remodeling after MI without increasing the frequency of arrhythmias. Mechanistically, the hiPSC-EC exosomes notably rescued the protein expression and function of the sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA-2a) and ryanodine receptor 2 (RyR-2) to maintain intracellular Ca2+ homeostasis and increase cardiomyocyte contraction after MI. The microRNA sequencing showed that miR-100-5p was the most abundant microRNA in exosomes. miR-100-5p could target protein phosphatase 1β (PP-1β) to enhance phospholamban (PLB) phosphorylation at Ser16 and subsequent SERCA activity, which contributes to the hiPSC-EC exosome-exerted cytoprotective effects on maintaining intracellular Ca2+ homeostasis and promoting cardiomyocyte survival.ConclusionThe hiPSC-EC exosomes maintain cardiomyocyte Ca2+ homeostasis to improve myocardial recovery after MI, which may provide an acellular therapeutic option for myocardial injury.

Project description:AimsThe effectiveness of cell-based treatments for regenerative myocardial therapy is limited by low rates of cell engraftment. Y-27632 inhibits Rho-associated protein kinase (ROCK), which regulates the cytoskeletal changes associated with cell adhesion, and has been used to protect cultured cells during their passaging. Here, we investigated whether preconditioning of cardiomyocytes, derived from human-induced pluripotent stem cells (hiPSC-CM), with Y-27632 improves their survival and engraftment in a murine model of acute myocardial infarction (MI).Methods and resultsAfter MI induction, mice were subjected to intramyocardial injections of phosphate-buffered saline, hiPSC-CM cultured under standard conditions (hiPSC-CM-RI), or Y-27632-preconditioned hiPSC-CM (hiPSC-CM+RI). The resulting engraftment rate calculated 4?weeks after implantation was significantly higher and the abundance of apoptotic transplanted cells was significantly lower in hiPSC-CM+RI recipients than in hiPSC-CM-RI animals. In cultured hiPSC-CM, Y-27632-preconditioning reversibly reduced contractile activity and the expression of troponin genes, while increasing their attachment to an underlying mouse cardiomyocyte (HL1) monolayer. Y-27632 preconditioning also increased the expression of N-cadherin and integrin ß1, the two cell junction proteins. hiPSC-CM+RI were also larger in cell area with greater cytoskeletal alignment and a more rod-like shape than hiPSC-CM-RI, both after transplantation (in vivo) and in culture. The effects of Y-27632 preconditioning on contractile activity and morphology of hiPSC-CMs in culture, as well as on their engraftment rate and apoptotic death in MI mouse grafts, could be recapitulated by hiPSC-CM treatment with the L-type calcium-channel blocker verapamil.ConclusionPreconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.

Project description:BACKGROUND:Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. METHODS:In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. RESULTS:The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. CONCLUSIONS:We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.

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Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

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Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

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