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

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.

The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

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.

Frontiers in Tissue Engineering is a carefully edited compilation of state-of-the-art contributions from an international authorship of experts in the diverse subjects that make up tissue engineering. A broad representation of the medical, scientific, industrial and regulatory community is detailed in the book. The work is an authoritative and comprehensive reference source for scientists and clinicians working in this emerging field. The book is divided into three parts: fundamentals and methods of tissue engineering, tissue engineering applied to specialised tissues, and tissue engineering applied to organs. The text offers many novel approaches, including a detailed coverage of cell-tissue interactions at cellular and molecular levels; cell-tissue surface, biochemical, and mechanical environments; biomaterials; engineering design; tissue-organ function; new approaches to tissue-organ regeneration and replacement of function; ethical considerations of tissue engineering; and government regulation of tissue-engineered products.

This book on cardiac extracellular matrix (ECM) features three sections, Fundamental Science, Pre-Clinical and Translational Science, and Clinical Applications. In the Fundamental Science section, we will cover the spectrum of basic ECM science from ECM’s role in development, biomechanical properties, cardiac ECM influence of cardiomyocyte biology, pathophysiology of ECM in heart disease, and ECM in tissue engineering. Section two, Preclinical and Translational Science, will discuss cardiac ECM technologies in the clinical pipeline including approaches to ECM as a therapeutic, animal models of cardiac research, tracking and imaging methods of cardiac ECM, and cGMP manufacturing and regulatory considerations for ECM based therapeutics. Finally, the third section, Clinical Applications, will highlight the clinical experience around cardiac ECM including therapeutic strategies targeting scar tissue in the heart, Clinical trial design and regulatory considerations, current human clinical trials in cardiovascular medicine and the role of pharmaceutical and biotech companies in the commercialization of ECM technologies for cardiovascular indications. This book provides a comprehensive review for basic and translational researchers as well as clinical practitioners and those involved in commercialization, regulatory and entrepreneurial activities.