Abstract
The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity.
Highlights
Drug associated cardiovascular toxicity causes severe morbidity (Schimmel et al, 2004) and is a major reason for attrition during drug discovery and development (Abassi et al, 2012; Albini et al, 2010; Laverty et al, 2011)
Cardiac fibroblasts serve a structural role by providing the extracellular matrix of the heart, mechanotransductive cues and paracrine factors that regulate cardiomyocyte maturation (Bowers et al, 2010; Porter and Turner, 2009; Souders et al, 2009), whilst cardiac endothelial cells form the myocardial microvasculature, which regulates the supply of oxygen and free fatty acids to the CMs (Aird, 2007)
All primary cells were detached with pre-warmed Accutase (Sigma, A6964) for 5 min at 37C, 5% CO2, centrifuged for 3 min at 1200 Â g before re-suspension in endothelial basal medium MV2 (PromoCell, C-22221) (supplemented with 5% foetal calf serum (FCS), epidermal growth factor (EGF; 5 ng mlÀ1), basic fibroblast growth factor, insulin-like growth factor 1 (IGF-1; 20 ng mlÀ1), vascular endothelial growth factor-A (VEGFA; 0.5 ng mlÀ1), Ascorbic Acid (1 lg mlÀ1) and Hydrocortisone (0.2 lg mlÀ1)
Summary
Drug associated cardiovascular toxicity causes severe morbidity (Schimmel et al, 2004) and is a major reason for attrition during drug discovery and development (Abassi et al, 2012; Albini et al, 2010; Laverty et al, 2011). Tissue engineering approaches offer great potential for developing 3D models of heart muscle for use in drug discovery and development and as cardiac tissue replacement in the clinic. These approaches include scaffold-free methods such as microtissues derived from hanging drops or in hydrogels and monolayer derived cell sheets or patches, and scaffold dependent methods (Hirt et al, 2014). Compared to all other cell combinations, spheroid microtissues derived from all 3 cardiac cell types showed remarkably enhanced contractile function in response to inotropic drugs, which suggested a greater degree of cardiomyocyte maturity. We conclude that noncardiomyocyte cells are critical for promoting an authentic cardiac phenotype in complex in vitro models and that such models can be used for more accurate drug safety screening and drug development
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
