Original Pre-Clinical Science
Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell–derived cardiomyocytes

https://doi.org/10.1016/j.healun.2017.01.001Get rights and content

Background

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes.

Methods

We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4+ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red–detected calcium transients and computer-assisted image analysis.

Results

After 8 days in culture, 94% ± 6% of the NKX2-5GFP+ cells were beating when hESCs embryonic bodies were plated on E4+ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP+ cardiomyocytes were close to the E4+ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges.

Conclusions

The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony.

Section snippets

Maintenance of hESC lines

The hESC cell lines with a green fluorescent protein (GFP) reporter for the cardiac gene, Nkx2.5, were obtained as previously described (NKX2-5GFP hESC).9 The hESC cell lines ES4 were purchased from Wicell Research Institute. The permissions for use of these cell lines were obtained after review by the Cornell-Rockefeller-Sloan Kettering Institute ESC Research Oversight Committee.

The hESCs were grown on feeder layer free conditions on growth factor-reduced BD Matrigel (#354230, BD Biosciences)

Cardiac differentiation optimization

We first used classical 2D cultures to derive cardiomyocytes from hESCs with or without an ECs feeder layer. The presence of endothelium induced increased differentiation (13.5% ± 1.5% in the control vs 18.6% ± 1.2%) with the ECs, as defined by the number of differentiated hESCs expressing NKX2-5GFP over the total number of hESCs as determined by flow cytometry (p < 0.01; Supplementary Figure 1A–C, available online at www.jhltonline.org); however, the efficiency of this protocol was quite low,

Discussion

Here, we demonstrated that the in vitro use of an activated endothelial feeder increased the differentiation of cardiomyocytes from hESCs and resulted in a differentiated cardiomyocyte population with a synchronous beating pattern that may be more adapted to clinical use.32, 33

Disclosure statement

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.

The authors thank the Flow Cytometry Facility within the Microscopy Core at Weill Cornell Medical College in Qatar for contributing to these studies. The Core is supported by the “Biomedical Research Program at Weill Cornell Medical College in Qatar," a program funded by Qatar Foundation. The authors acknowledge

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    These authors contributed equally to this work.

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