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Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues

ABSTRACT: Cardiac in vitro models have become increasingly easy and affordable with the optimisation of stem cell-based cardiomyocyte (CM) differentiation. However, the CMs obtained are still far from being as mature as their in vivo counterparts. Here we study the cellular phenotype of human pluripotent stem cell-derived CMs by comparing the single-cell level gene expression profiles among different engineered cardiac tissue configurations: human ventricular cardiac anisotropic sheet, human ventricular cardiac tissue strip, and human ventricular cardiac organoid chamber (hvCOC). As a control, the spontaneously formed 3D cardiac spheroid aggregates (CS) were used. Our results suggest that the level of maturation increases with the level of complexity of the engineered tissues with CS being the least mature and hvCOC the most mature tissue. We show that the contractile components are the first function to mature, followed by electrophysiology and oxidative metabolism. Notably, the cellular structures in the 2D constructs show higher organisation while metabolic maturity preferentially increase in the 3D structures. We conclude that more complex tissue engineering models could offer more physiological and reliable in vitro matured cardiac tissue model for drug screening or disease modelling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200277 | GEO | 2023/03/08

REPOSITORIES: GEO

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Project description:Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23±4.69%, ViPSC-CMs: 90.25±4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes to be responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. ¬ Conclusions: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

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Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues

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