Enhancement of human iPSC-derived cardiomyocyte maturation by chemical conditioning in a 3D environment

https://doi.org/10.1016/j.yjmcc.2019.10.001Get rights and content

Highlights

  • 3D microtissues used to probe biochemical maturation of hiPSC-cardiomyocytes (CMs).

  • Thyroid hormone, dexamethasone, and IGF-1 (TDI) improve CM maturation in 3D.

  • Gene expression and ultrastructure show improved adult cardiac phenotype.

  • Enhanced electrophysiological properties, contraction and ionotropic response

Abstract

Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.

Introduction

Induced pluripotent stem cells (iPSCs) can self-renew and differentiate into all cell types, and thus provide a potentially unlimited ex vivo source of cells and tissue for regenerative medicine, disease modeling and drug discovery. Such a cell source is especially important for cell types, such as cardiomyocytes (CMs), that have limited regenerative capacity, and also exhibit significant loss due to ageing or disease. However, human iPSC-derived cardiomyocytes (hiPSC-CMs) typically display fetal-like structure [1,2] and metabolism [3,4], along with underdeveloped excitation–contraction coupling, calcium handling, and electrophysiological properties [[5], [6], [7]]. This relatively immature state compared to adult myocardium is an important factor that must be overcome to enable many of the potential applications of hiPSC-CMs [8,9].

To improve the degree of maturation of hiPSC-CMs, different approaches have been employed including three-dimensional (3D) culture methods [[10], [11], [12], [13], [14], [15], [16], [17]], electrical and mechanical stimulation [[10], [11], [12],17,18], culturing cells on extracellular matrices mimicking the in-vivo environment [13,19], in vivo engraftment [20], or prolonged growth in vitro [21]. Strategies involving biochemical treatments, such as triiodothyronine [22], dexamethasone [23], or insulin-like growth factor-1 (IGF-1) [14,24], HIF-1α inhibitors, PPARα agonists [25] alone or in combination [15,26,27], have been shown to enhance the electrophysiological properties, bioenergetics, and contractile force generation of hiPSC-CMs. Notably, Birket et al. [26] demonstrated that a combination of thyroid hormone, dexamethasone, and IGF-1 (TDI) promoted maturation of function in cultures of normal hiPSC-CMs and those with mutations in contractile proteins. TDI has been used to enhance hiPSC-CMs metabolic maturation, in combination with other factors in cardiac spheroids [25]. We posit that recreation of the circumstances required for tissue maturation requires not only biochemical signals, but also the cellular interactions and mechanical loading that are present in 3D models that better recapitulate the native tissue environment.

In this study, we used the same optimized biochemical intervention (TDI) reported previously [26] in 3D cardiac microtissues (CMTs) grown on a microfabricated tissue gauge (μTUG) platform [[28], [29], [30], [31], [32]] to investigate systematically transcriptional, structural and functional maturation. We found that TDI treatment altered the transcriptional profile, generated cells with a more ventricular cardiomyocyte (vCM)-like electrophysiological and Ca2+ handling phenotype and enhanced the tissue structure/ultrastructure and contractile performance of 3D CMTs.

Section snippets

Human iPSC cardiomyocyte generation and defined chemical treatment

Human iPSCs were created under the auspices of a Johns Hopkins IRB approval. The iPSCs were generated from peripheral blood mononuclear cells (PBMCs) using Sendai virus [33] containing Yamanaka factors for reprogramming. hiPSCs were maintained in E8 culture medium (A1517001, Gibco) and differentiated into CMs via small-molecule modulation of Wnt signaling [34]. Briefly, four days prior to differentiation, hiPSCs were seeded on Geltrex (A1413202, Gibco) coated plates. When cells reached 80–90%

Structural and ultrastructural characteristics of hiPSCs-CMs in monolayers and CMTs

Our differentiation protocol (Fig. 1A) yielded hiPSC-derived cell populations composed of ~90% TNNT2+ hiPSC-CMs with <10% CD90+ cells, as confirmed by flow cytometry at Day 13–15 (Supplementary Fig. S2). Following treatment with TDI for 7 days (Day 19–21), monolayer cultures of hiPSC-CMs showed significant morphological changes as illustrated in Fig. 1B. Phase contrast images (Supplementary Figs. S3A and S3B, and Supplementary Video S1) showed that the cells had a greater length to width ratio

Discussion

In this study we have found that hiPSC-CMs both in 2D culture and in CMTs show structural and functional changes after TDI treatment that make them more closely resemble working vCMs and myocardium. Structurally, the TDI-treated CMs were larger, more rod-shaped, and better aligned along their long axes with longer sarcomere lengths. Ultrastructural analysis confirmed that TDI treated monolayers and CMTs show highly ordered sarcomeres, including dense myofibrils with H- and I-bands, gap

Conclusion

Treatment with TDI has significantly improved the dynamic mechanical properties of 3D hiPSC cardiac microtissues. This, together with the enhanced functional properties that this maturation approach yields, has the potential to yield improved model systems that can advance both mechanistic studies and the development of new therapies for the treatment of cardiac diseases.

Disclosures

None.

Acknowledgements

This research was supported in part by the Maryland Stem Cell Research Fund (2016-MSCRFI-2735), the Zegar Family Foundation, and the Magic that Matters Fund. Confocal microscopy was carried out at the Johns Hopkins University School of Medicine's Microscope Facility, which is supported by NIH grant S10OD-016374. Flow cytometry experiments were performed at the Homewood Flow Cytometry Resource in the Johns Hopkins University Integrated Imaging Center, which is supported by the Johns Hopkins

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    Present address: Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, 1300 Morris Park Ave, Belfer 312, Bronx, NY 10461, USA.

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