Facilitated maturation of cardiomyocytes derived from human embryonic stem cells in 3D collagen matrix upon mesenchymal cell supplementation and mechanical stretch

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as promising cell source for regenerative medicine, drug testing and disease modeling. Nevertheless, these cardiomyocytes are immature in terms of contractile structure, metabolism and electrophysiological properties. There are increasing efforts using biological, chemical and physical approaches to facilitate maturation of hESC-CMs, with 3D matrix recognized as an optimal in vitro platform. In light of the previous findings, cardiac tissue strips were fabricated by encapsulating hESC-CMs into collagen/matrigel matrix in current study. The engineered tissue strips contract against mounted ends and grow into compact tissues with spontaneous beating. We hypothesize that addition of mesenchymal cells in small amount could accelerate maturation of hESC-CMs in collagen matrix, with mechanical stretch assumed to be superior to static stress in driving hESC-CM maturation. More specifically, we aim to demonstrate functional improvements of engineered cardiac tissue strips in terms of structural arrangement, mechanical properties, contractile performance and gene expression. Results showed that supplementation of mesenchymal cells at 3% could already boost maturation of fabricated heart tissue strips, where benefits of mesenchymal stem cell addition were shown to be comparable to that of fibroblast. Both cell types significantly promoted compaction and cell spreading to the same extent, with similar molecular signature in terms of gene expression and protein localization shown at tissue level. hMSC co-encapsulated tissues possess greater mechanical properties than hFB added counterparts such as elastic modulus, passive tension and twitch force under strain, yet the difference was not significant. Cyclic stretch was demonstrated to render better maturated engineered cardiac tissues when comparing with static stress, with static stretch showed similar advantages, albeit to a lesser extent. Both stretch schemes outperformed static stressed samples, as evidenced by more elongated sarcomere, stronger twitch force, steeper stress-strain curve, greater elastic modulus and better expression of major contractile and hypertrophic genes. However, statistical significance was achieved only between cyclic stretched tissue strips and static stressed group in most of the evaluation assays, suggesting superiority of the cyclic stretch in functionalizing engineered cardiac tissue. In vitro maturation of cardiomyocytes is a complex process, which could be achieved through combination of multiple approaches such as mechanical loading, electrical stimulation, niche cell addition and perfusion. This study proved that mesenchymal stem cells could be considered equivalent to fibroblasts in facilitating maturation of hESC-CMs within 3D collagen matrix. Moreover, mode of loading does affect functionality of engineered cardiac tissue, with cyclic stretch demonstrated to elicit greatest improvement. Findings of current study contribute to bioengineering of functional heart tissue from hESC-derived cardiomyocytes in the long run.published_or_final_versionMechanical EngineeringDoctoralDoctor of Philosoph