Biochemical and Biophysical Research Communications
Creation of human cardiac cell sheets using pluripotent stem cells
Highlights
► Bioreactor-based suspension culture is suitable for the cardiac induction of human iPS cells. ► Human iPS cells-derived cardiac cell sheets showed the spontaneous pulsation. ► The electrophysiological connections are observed between human cardiac cell sheets.
Introduction
Regenerative medicine is thought to be a promising therapeutic strategy for the treatment of severe heart failure. We previously developed an original scaffold-free tissue engineering technology, designated as “cell sheet-based tissue engineering”, using temperature-responsive culture dishes covalently bonded to the temperature-responsive polymer poly(N-isopropylacrylamide) [1]. Lowering the culture temperature promotes a rapid surface transition from hydrophobic to hydrophilic, which enables collection of a viable monolayer cell sheet with full preservation of the cell-cell contacts and extracellular matrices [2]. Many studies have shown that cell sheet-based bioengineered tissue transplantation improves the cardiac function of various types of heart failure models [3], [4], [5]. However, recent evidences have suggested that paracrine mechanisms, including angiogenesis and cardioprotection mediated by the secreted factors of transplanted cells, mainly contribute to the improved cardiac function [3], [6]. Furthermore, an adult human heart is reported to contain over a billion cardiomyocytes [7], indicating that creation of bioengineered thickened cardiac tissue in vitro, which directly contributes to contraction when transplanted, might be a prerequisite. Previously, we reported the development of cell-dense 1 mm thick cardiac tissue by repeated transplantation of triple-layered neonatal rat cardiac cell sheets [8]. Recently, we have also reported the development of cardiac cell sheets derived from mouse embryonic stem cells (ESCs) after three-dimensional suspension culture [9]. Mouse ESC-derived cardiac cell sheets have similar electrophysiological properties to those of neonatal rat cardiomyocytes, indicating that layered stem cell-derived cardiac cell sheets might show synchronous contraction. However, it remains unknown whether human pluripotent stem cell-derived cardiomyocytes are suitable for creating cell sheets in terms of their electrophysiological functions.
Many recent reports have suggested that human pluripotent stem cells, including ESCs and induced pluripotent stem cells (hiPSCs), differentiate into cardiomyocytes through embryoid body (EB) formation [10], [11] and monolayer culture [12], [13]. Although suspension culture of EBs is easy in terms of scale-up, advancements to overcome the limitation of EB size heterogeneity for efficient cardiac differentiation might be necessary. Conversely, the monolayer-based method is able to consistently produce cardiomyocytes with high efficacy, but scale-up might be a significant challenge. Recent methodological progress has enabled production of >80% cardiomyocytes in both methods [14], [15]. However, the differences in cardiomyocyte properties from the viewpoint of cell sheet-based tissue engineering remain elusive.
The aims of this study were to establish easy and effective methods for collecting cardiomyocytes derived from hiPSCs to create cardiac cell sheets, and to elucidate the electrophysiological functions of hiPSC-derived cardiac cell sheets.
Section snippets
Antibodies
The following antibodies were used for immunocytochemistry and/or flow cytometry: anti-sarcomeric α-actinin (Sigma–Aldrich, St. Louis, MO), anti-cardiac troponin T (cTnT; Thermo Scientific, Rockford, IL), anti-CD31 (BD Bioscience, San Jose, CA) and anti-Tra-1 60 (Millipore, Billerica, MA) mouse monoclonal antibodies, anti-SM22 (Abcam, Cambridge, UK), anti-connexin 43 (Enzo Life Sciences, Farmingdale, NY) and anti-von Willebrand factor (vWF, Dako, Japan) rabbit polyclonal antibodies, and an
Cardiac differentiation of hiPSCs
Various types of methods to induce cardiac differentiation of human pluripotent stem cells have been reported [10], [11], [12]. In this study, we used two types of methods; (1) suspension culture in the bioreactor, and (2) monolayer culture. Small aggregates of hiPSCs (approximately 2 × 107 cells) were seeded in the bioreactor containing mTeSR1 medium, resulting in the formation of many EBs, and the cell number increased up to around 4 × 107 cells at day 3 (Fig. 2A and B). Then, EBs were treated
Discussion
The present study shows that a bioreactor-based method sufficiently induces cardiac differentiation of around 80% of hiPSCs. Cardiomyocytes in the cell sheets showed spontaneous and synchronous beating, and the cell sheet itself showed macroscopic contraction. Furthermore, electrophysiological connections were observed between cardiac cell sheets. Therefore, human pluripotent stem cell-derived cardiac cell sheets might be used to construct functional thickened cardiac tissue.
Many reports have
Acknowledgments
This research was funded by a Grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP).
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