Abstract 2279: Fibroblasts Are Essential for the Generation of Embryonic Stem Cell Derived Bioengineered Myocardium
The heart is mainly composed of non-myocytes (NMs; ~70%). Here we used a novel in vitro model of heart muscle development, namely engineered heart tissue (EHT) to test the hypothesis that NMs are essential for cardiogenesis.
Methods: Murine embryonic stem cell-derived cardiomyocytes (αMHC-NeoR) were generated in bioreactor cultures under G418-selection. Cardiomyocyte (CM) functionality and purity were assessed by action potential (AP) recordings. EHTs were generated from differentiated CMs containing 0 –50% cardiac NMs (1.5×106 cells/EHT), matrigel, and collagen (volume: 450 μl). At later stages of EHT-culture G418 (200 μg/ml) was added to control NM growth. EHTs were characterized by isometric force measurements, morphological, and gene expression analyses (gene-array, qPCR).
Results: 24±7×106 CMs could be generated in a single 500 ml bioreactor run (n=3). AP-recordings (n=95) revealed the presence of ventricle- (n=24), Purkinje- (n=31), atrial-(n=11), and pacemaker-like (n=17) CMs, and 7 cells with intermediate AP kinetics, as well as 5 cells without APs. Coherently beating EHTs could not be generated from purified myocytes alone. In contrast, NMs supplementation (25%) facilitated the formation of force-generating EHT. The latter displayed positive inotropic effects under increasing extracellular Ca2+ (0.4 to 2.4 mM: 18±4 vs. 64±13 μN; n=10) and addition of isoprenaline (1 μM: 37±8 vs.46±8 μN; n=10) as expected for normal heart muscle. Histological analysis revealed the formation of thick cTnI-, f-actin-, and Cx43-positive muscle bundles within EHTs. After G418 selection (EHT culture days 3– 6) EHTs showed abundant expression of CM (αMHC) and fibroblast (DDR2) specific transcripts (n=4); smooth muscle (SM-MHC) and endothelial (Tie2) cell markers were only detected at minimal amounts. In addition, gene expression analyses revealed strong extracellular-matrix (ECM) synthesis (collagen1-α1 and -α2) likely by fibroblasts (n=4).
Conclusion: Our data suggest that fibroblasts are essential for in vitro cardiogenesis and likely maintain the ECM in EHT. Whether the observed fibroblast-mediated cardiogenic effects are mediated by direct cell-cell interactions, paracrine factors, or biomechanical stimuli remains to be elucidated.