Cardiac tissue engineering enables the generation of functional human cardiac tissue using cells in combination with biocompatible materials. Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electrical pacing on the maturation of hPSC-derived cardiac tissues. In the first part of the study, cardiomyocytes derived from human induced pluripotent stem cells (hIPSCs) were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subject to different stress and electrical pacing conditions. This engineered human myocardium exhibits Frank-Starling curve-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated at least 10-fold increase in contractility and tensile stiffness, greater cell alignment, and a 1.5-fold increase in cell size and cell volume fraction within the constructs. Stress conditioning also increased sarco-endoplasmic reticulum calcium transport ATPase 2 (SERCA2) expression. When electrical pacing was combined with static stress conditioning, the tissues showed an additional 2-fold increase in force production, tensile stiffness, and contractility, with no change in cell alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 and SERCA2 further increased by combined static stress and electrical stimulation. These studies demonstrate that electrical pacing and mechanical stimulation promote both the structural and functional maturation of hiPSC-derived cardiac tissues. In the second part of the study, cardiovascular progenitor (CVP) cells derived from hPSC were used as the input cell population to generate engineered tissues. The effects of a 3-D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue were evaluated. Compared to 2-D culture, the unstressed 3-D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3-D culture suppressed smooth muscle cell maturation. Mechanical stress conditioning further improved cardiomyocyte maturation. Cyclic stress-conditioning increased expression of several cardiac markers, like beta-myosin and cTnT, and the tissue showed enhanced force production. This 3-D system has facilitated understanding of the effect of mechanical stress on the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissues. In conclusion, we were able to create a complex engineered human cardiac tissue with both stem cell-derived cardiomyocytes and CVP cells. We showed that how environmental stimulations like mechanical stress, electrical pacing, and 3-D culturing can affect the maturation and specification of cells within the engineered cardiac tissues. The study paves our way to further apply these engineered cardiac tissues to other in vitro and in vivo usages like drug testing, clinical translation, and disease modeling.

Cardiac Tissue Engineering: Methods and Protocols presents a collection of protocols on cardiac tissue engineering from pioneering and leading researchers around the globe. These include methods and protocols for cell preparation, biomaterial preparation, cell seeding, and cultivation in various systems. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Cardiac Tissue Engineering: Methods and Protocols highlights the major techniques, both experimental and computational, for the study of cardiovascular tissue engineering.

Stem Cells in Clinical Practice and Tissue Engineering is a concise book on applied methods of stem cell differentiation and optimization using tissue engineering methods. These methods offer immediate use in clinical regenerative medicine. The present volume will serve the purpose of applied stem cell differentiation optimization methods in clinical research projects, as well as be useful to relatively experienced stem cell scientists and clinicians who might wish to develop their stem cell clinical centers or research labs further. Chapters are arranged in the order of basic concepts of stem cell differentiation, clinical applications of pluripotent stem cells in skin, cardiac, bone, dental, obesity centers, followed by tissue engineering, new materials used, and overall evaluation with their permitted legal status.

This dissertation, "Facilitated Maturation of Cardiomyocytes Derived From Human Embryonic Stem Cells in 3D Collagen Matrix Upon Mesenchymal Cell Supplementation and Mechanical Stretch" by Wei, Alvin, Zhang, 張偉, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: 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. DOI: 10.5353/th_b5689289 Subjects: Heart cells Embryonic stem cells