Single-cell analysis reveals the purification and maturation effects of glucose starvation in hiPSC-CMs

https://doi.org/10.1016/j.bbrc.2020.11.076Get rights and content

Highlights

  • ScRNA-Seq analysis was performed for hiPSC-CMs subjected to glucose starvation.

  • Glucose starvation increases the subpopulation of cardiomyocytes in the late stage.

  • Glucose starvation improves the feasibility of hiPSC-CMs application.

Abstract

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) play a critical role in most translational and clinical applications. Although glucose starvation (GS) has been evaluated during cellular purification, there has been no comprehensive evaluation of the transcriptional heterogeneity of these cells. Here, we applied GS for 3 days starting at day 10 of differentiation, and then, harvested hiPSC-CMs at day 20 for single-cell RNA sequencing (scRNA-seq). We found that GS dramatically reduced the proportion of non-cardiomyocytes cells and increased the number of late-stage cardiomyocytes. We also recorded an increase in the expression of MYH6, MYH7, ACTN2, TNNT2, and several other genes associated with the structural and functional maturation of cardiomyocytes. Further analysis indicated that these changes were focused on the signaling pathways involved in the regulation of the actin cytoskeleton, cardiac muscle development, and cardiac muscle contraction. Finally, pseudotime analysis revealed that GS hiPSC-CMs developed in a more mature direction. Together, these results suggest that GS treatment improves the purity and maturation of hiPSC-CMs, which should increase the feasibility of hiPSC-CMs applications.

Introduction

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) play a critical role in disease modeling, drug screening, toxicity evaluations, and the development of cell-based therapies for damaged heart [[1], [2], [3], [4]]. These cells were first derived using the embryoid body (EB)-based differentiation method, which relied on the application of the growth factors transforming growth factor β (TGF-β) and bone morphogenetic protein 4 (BMP-4) [5,6]. Although effective, this was not an efficient protocol, and further analysis revealed that modulating Wnt/β-catenin signaling using small molecules improves differentiation efficiency [7]. However, the hiPSC-CMs produced using this protocol were still immature and included a variety of cellular subtypes, including atrial-like, ventricular-like, sinus-like, residual undifferentiated cells, and other cell lineages, which may reduce the reliability of these hiPSC-CMs in certain applications such as drug screening [8,9]. Therefore, improving differentiation efficiency, reducing heterogeneity, and increasing maturity are key factors in advancing the application of hiPSC-CMs.

In recent years, glucose starvation (GS) has been adopted to improve purity [10] although the specifics of the subpopulations of hiPSC-CMs isolated using this method have not been described. Single-cell RNA sequencing (scRNA-seq) has played an essential role in revealing cell types, determining cell subpopulations, accurately reflecting the heterogeneity of cellular populations, and describing cell fate lineages [11]. Here, we performed scRNA sequencing to explore the effect of GS on hiPSC-CMs differentiation.

Section snippets

Cardiomyocyte differentiation from hiPSCs

The hiPSCs line (HNF–P30–P11) used in this study was obtained from OSINGLAY, China. We used the classical cardiomyocyte differentiation protocol, GiWi, which uses small molecule GSK3 and Wnt inhibitors to differentiate hiPSC-CMs [7]. Briefly, when the hiPSCs colonies reached 80%–90% confluence they were dissociated into single-cell suspensions using versene solution (15040066, Gibco, USA) and resuspended in hiPSCs maintenance medium. On day 1, cells were cultured in RPMI/B-27 without insulin

HiPSCs characterization

To verify the pluripotency of the hiPSCs, the morphology of the cells was first observed using an optical microscope, and pluripotent markers were confirmed by IF and qPCR. Under magnification, the hiPSCs were densely packed with a high nucleus-plasma ratio and a uniform cell shape (Supplementary Fig. 1A), and the qPCR analysis indicated that these cells all expressed higher levels of the pluripotency markers OCT4, SOX2, and NANOG compared with the human cardiac fibroblast cells used as the

Discussion

There are several reports available for enriched cardiomyocytes population by GS but the transcriptional heterogeneity of these cells has been not been evaluated comprehensively [10]. Here we performed scRNA-seq to characterize hiPSC-CMs and identified 15 unique transcriptome profiles, which could be further divided into five cell types based on their expression of specific cardiac differentiation markers [16,17]. We found that the proportion of non-cardiomyocytes decreased dramatically

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Collaborative Innovation Center for the Prevention and Treatment of Cardiovascular Diseases in Sichuan Province [grant no. xtcx2016-14] and by the National Natural Science Foundation of China [grant no. 81870261].

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    These authors contributed equally to this work.

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