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Improving Generation of Cardiac Organoids from Human Pluripotent Stem Cells Using the Aurora Kinase Inhibitor ZM447439.

ABSTRACT: Drug-induced cardiotoxicity reduces the success rates of drug development. Thus, the limitations of current evaluation methods must be addressed. Human cardiac organoids (hCOs) derived from induced pluripotent stem cells (hiPSCs) are useful as an advanced drug-testing model; they demonstrate similar electrophysiological functionality and drug reactivity as the heart. How-ever, similar to other organoid models, they have immature characteristics compared to adult hearts, and exhibit batch-to-batch variation. As the cell cycle is important for the mesodermal differentiation of stem cells, we examined the effect of ZM447439, an aurora kinase inhibitor that regulates the cell cycle, on cardiogenic differentiation. We determined the optimal concentration and timing of ZM447439 for the differentiation of hCOs from hiPSCs and developed a novel protocol for efficiently and reproducibly generating beating hCOs with improved electrophysiological functionality, contractility, and yield. We validated their maturity through electro-physiological- and image-based functional assays and gene profiling with next-generation sequencing, and then applied these cells to multi-electrode array platforms to monitor the cardio-toxicity of drugs related to cardiac arrhythmia; the results confirmed the drug reactivity of hCOs. These findings may enable determination of the regulatory mechanism of cell cycles underlying the generation of iPSC-derived hCOs, providing a valuable drug testing platform.

SUBMITTER: Lee SJ

PROVIDER: S-EPMC8698385 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.MethodsThe peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.ResultsCD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

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Improving Generation of Cardiac Organoids from Human Pluripotent Stem Cells Using the Aurora Kinase Inhibitor ZM447439.

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