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A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels.

ABSTRACT: Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cells and reaggregated into smaller spheroids using the microfabricated vessels. This reaggregation process raised WNT5A and WNT11 expression levels and improved the quality of cardiomyocyte population compared to that in a control group in which dissociation and reaggregation were not performed.

SUBMITTER: Miwa T

PROVIDER: S-EPMC7256449 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels.

Miwa Tatsuaki T Idiris Alimjan A Kumagai Hiromichi H

Regenerative therapy 20200526

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cell ...[more]

PMID: 32490063

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Project description:Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

Dataset Information

A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels.

Publications

A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels.

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