Research Article: A net-shaped multicellular formation facilitates the maturation of hPSC-derived cardiomyocytes through mechanical and electrophysiological stimuli

Date Published: April 14, 2018

Publisher: Impact Journals

Author(s): Taoyan Liu, Chengwu Huang, Hongxia Li, Fujian Wu, Jianwen Luo, Wenjing Lu, Feng Lan.


The use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Although many approaches have been reported to improve the maturation of hiPSC-CMs, the elucidation of the process of maturation is crucial. We applied a small-molecule-based differentiation method to generate cardiomyocytes (CMs) with multiple aggregation forms. The motion analysis revealed significant physical differences in the differently shaped CMs, and the net-shaped CMs had larger motion amplitudes and faster velocities than the sheet-shaped CMs. The net-shaped CMs displayed accelerated maturation at the transcriptional level and were more similar to CMs with a prolonged culture time (30 days) than to sheet-d15. Ion channel genes and gap junction proteins were up-regulated in net-shaped CMs, indicating that robust contraction was coupled with enhanced ion channel and connexin expression. The net-shaped CMs also displayed improved myofibril ultrastructure under transmission electron microscopy. In conclusion, different multicellular hPSC-CM structures, such as the net-shaped pattern, are formed using the conditioned induction method, providing a useful tool to improve cardiac maturation.

Partial Text

Cardiovascular disease is currently the leading cause of death worldwide. Despite an increased effort toward basic research and drug discovery, many cardiovascular diseases still have no curative treatments. As a type of terminally differentiated cells, the application of cardiomyocytes (CMs) is hindered by the difficulties in obtaining human cardiac tissue and the inability to propagate heart samples in culture. Therefore, most existing research has utilized animal models to mimic human heart disease. However, critical physiological and biochemical differences exist between the animal and human heart, especially for CMs. Fortunately, human-induced pluripotent stem cell (hiPSC) technology plays a vital role in the advancement of cardiovascular research and medicine and includes refined protocols for hiPSC reprogramming and cardiac differentiation that enable the derivation of human CMs with patient-specific phenotypes. This technology shows great potential in regenerative therapy and mechanistic investigations of cardiac diseases [1]. Previously, several hereditary cardiomyopathies were modeled in vitro using hiPSC technology, including hypertrophic cardiomyopathy (HCM) [2], dilated cardiomyopathy (DCM), long QT syndrome (LQT) [3] and LEOPARD syndrome . However, these in vitro-derived hiPSC-CMs are generally immature, with features resembling fetal CMs rather than adult myocardial tissue [4,5].

In this study, we revealed that differently shaped hiPSC-CM populations from the same differentiation batch exhibit different cellular and molecular properties. Specifically, the net-shaped CMs were more mature than the sheet-shaped cells, suggesting that the physical formation of the multicellular structure could significantly affect the maturation of hiPSC-CMs. Even within the same culture, CMs exhibit various developmental or maturation statuses due to the different types of aggregates formed, which may lead to variations in gene expression profiles and contractile and ultrastructure properties. The generation of multicellular formation patterns of hiPSC-CMs is a common phenomenon, but no previous reports have described the differences among these patterns. Our study is the first to analyze the movement of net-shaped CMs and sheet-shaped CMs, and this morphological arrangement of hiPSC-CMs exerted a profound impact on their maturation status. Three typical forms of CM clusters are readily obtained using the small molecule-based differentiation method: isolated beating clusters, net-shaped CMs and sheet-shaped CMs. The isolated beating clusters were derived from a lower efficiency differentiation process. We then focused on the more reproducible net-shaped and sheet-shaped CMs, which were derived from differentiation proceeding at much higher efficiencies (80-95% TNNT2+). The percentages of ventricular and atrial cells were not significantly different in cells with the two distinct morphologies (Figure S3). Interestingly, a lower seeding density (3.0×103 and 1.2×104 cells/cm2) of hPSCs resulted in highly efficient net-shaped CM production. Different seeding densities could lead to the growth patterns of mesoderm specification and the following cardiogenesis, which eventually lead to the variation of 3D structure. This simple and effective method was used to obtain hPSC-CMs with the preferred morphologies.




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