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.

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

Cellular and Molecular Pathobiology of Cardiovascular Disease focuses on the pathophysiology of common cardiovascular disease in the context of its underlying mechanisms and molecular biology. This book has been developed from the editors' experiences teaching an advanced cardiovascular pathology course for PhD trainees in the biomedical sciences, and trainees in cardiology, pathology, public health, and veterinary medicine. No other single text-reference combines clinical cardiology and cardiovascular pathology with enough molecular content for graduate students in both biomedical research and clinical departments. The text is complemented and supported by a rich variety of photomicrographs, diagrams of molecular relationships, and tables. It is uniquely useful to a wide audience of graduate students and post-doctoral fellows in areas from pathology to physiology, genetics, pharmacology, and more, as well as medical residents in pathology, laboratory medicine, internal medicine, cardiovascular surgery, and cardiology. - Explains how to identify cardiovascular pathologies and compare with normal physiology to aid research - Gives concise explanations of key issues and background reading suggestions - Covers molecular bases of diseases for better understanding of molecular events that precede or accompany the development of pathology

Native cardiac tissue is comprised of many different cell types that work cooperatively for proper tissue function. Combining self-assembled tissue engineering strategies that provide a fully-defined platform to study pairwise interactions between different cardiac cell types, with human pluripotent stem cell (hPSC) technologies, such as robust differentiation strategies and genome editing capabilities, has enabled our comprehensive studies of heterotypic interactions between cardiomyocytes and various non-myocyte sources in order to determine the specific contributions of non-myocytes to cardiac microtissue fate and function. We approached these studies in a systematic manner with narrowing focus.We first broadly tested the most commonly-used stromal cells in cardiac tissue engineering studies and found that the different sources of stromal cells (primary human-derived vs. stem cell-derived; from different types of primary tissues) were distinct in terms of their surface marker expression, morphometry, and gene expression. These differences carried over into their ability to support engineered cardiac tissue formation and function, where only primary human cardiac fibroblasts and primary human dermal fibroblasts paired with hPSC-cardiomyocytes resulted in microtissues with the most robust tissue self-assembly and advanced calcium handling function. Since the tissue-specific cardiac fibroblasts were able to positively support cardiac microtissue culture, we further characterized the specific contributions of different types of non-myocytes (endothelial cells, fetal human cardiac fibroblasts, adult human cardiac fibroblasts) in the context of heterotypic cardiac microtissue phenotype and function, at the single-cell and tissue-level, respectively. We found that 1 week after tissue formation, the cardiac microtissues containing the cardiac fibroblasts displayed more mature calcium handling properties relative to the tissues that contained endothelial cells and the tissues made from only cardiomyocytes, and that the cardiomyocytes paired with the cardiac fibroblasts were transcriptionally distinct from cardiomyocytes from the other tissues. However, after extended culture duration (1 month), the distinction between cardiac microtissues with cardiac fibroblasts versus without was lost, with the cardiomyocytes exhibiting similar transcriptomic profiles and the tissues displaying similar calcium transients. Furthermore, at both time points, there were no discernable differences between the different age cardiac fibroblasts, potentially because the source (isolated from primary tissue) was a bigger mismatch with stem cell-derived cardiomyocytes than the ontogenic difference.Inspired by the pairing of different technologies to assess single-cell-level phenotype in the context of microtissue-level function, we sought to further characterize individual cell properties within intact 3D microtissues in order to better link the single cell building blocks to tissue-level properties. We used light sheet fluorescence microscopy to quantify 3D heterotypic multicellular organization as well as identify individual cardiomyocyte functional heterogeneity within heterotypic cardiac microtissues. Overall, this study demonstrated that advanced imaging techniques can be a powerful tool to dissect complex heterotypic interactions without removing the cells from their 3D environment. Lastly, to dig deeper into the mechanisms governing the heterotypic interactions between cardiomyocyte and non-myocytes in our 3D engineered microtissues, we first had to generate cardiac tissues made from entirely stem cell-derived cellular constituents in order to take advantage of the robust genome engineering strategies developed for hPSCs. We were able to generate entirely-isogenic tissues when two differentiation protocols for the derivation of cardiac fibroblasts were published in 2019. We evaluated the different hPSC-cardiac fibroblast subtypes generated by these protocols in our heterotypic cardiac microtissue platform and found that they behaved similarly to one another and to microtissues made with primary human fetal cardiac fibroblasts in their ability to quickly self-assemble into tissues and their calcium handing function. We then knocked down one of the most-cited gap junctions that connects cardiomyocytes and cardiac fibroblasts in the heart, connexin 43, in the hPSC-derived cardiac fibroblasts using an inducible CRISPR interference method. Heterotypic cardiac microtissues generated with the knockdown fibroblasts displayed diminished calcium handling function, indicating a potential role for cardiac fibroblast support of cardiomyocyte function. Further mechanistic understanding of the interactions between cardiac cell populations can be determined in a similar manner, within the context of our fully-defined, tailored microtissue platform. Taken together, this body of work provides a basis for the study of multicellular heterotypic interactions in 3D engineered models of myocardial tissue from human pluripotent stem cells. Future studies can build on this work by generating more complex microtissue constructs (i.e. incorporating more than two cell types, or modulating the cell types) as well as modeling cardiac diseases in tissue format. Combining genome editing, advanced imaging, and next-generation sequencing technologies enables customizable generation and comprehensive characterization of the multicellular interactions within engineered heterotypic tissue constructs.