Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as exciting new tools in the field of cardiac research that possess immense therapeutic potential and can serve as innovative pre-clinical platforms for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Most prior efforts on improving hPSC-CM maturation have utilized cues stemming from the in vivo cardiac milieu. While these studies have resulted in modest hPSC-CM maturation improvements, individually they fail to fully recapitulate the adult cardiomyocyte phenotype. Thus, the field has gravitated towards the creation of more biomimetic microenvironments that have the ability to combine multiple signaling factors into one in vitro culture platform. Herein we provide a 2D in vitro substrate platform inspired by the myocardial microenvironment that improves hPSC-CM maturation while simultaneously allowing for quantitative measurements of mechanical and electrophysiological outputs. Substrate stiffness and micropatterned ECM are two known hPSC-CM pro-maturation cues. We study the effects of these parameters on hPSC-CMs and cardiac fibroblast generated from human induced pluripotent stem cells (hiPSC-CFs). Similar to our prior result on glass substrates, hPSC-CMs patterned on 10kPa PDMS align their internal cytoskeletal network in accordance with the micropatterned lanes. We mimic a functional cardiac syncytium by connecting micropatterned lanes with micropatterned bridges seeded with hPSC-CMs. hPSC-CMs patterned on this 15 degree chevron pattern displays anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the direction of the lanes compared to the transverse direction. hPSC-CFs cultured on micropatterned lanes and the 15 degree chevron pattern remodel the underlying ECM and produce fibers of collagen and fibronectin parallel to the feature direction. When co-cultured together on this pattern, ECM is produced by hPSC-CFs and hPSC-CMs display improved calcium kinetics and contractile strain. The ability to test factors individually and concomitantly in one biomimetic platform will lend new insights and aid in the directed maturation of immature hPSC-CMs, ultimately furthering our basic understanding of cardiac biology and providing novel platforms for drug discovery and toxicity testing.

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.

Myocardial tissue engineering (MTE), a concept that intends to prolong patients’ life after cardiac damage by supporting or restoring heart function, is continuously improving. Common MTE strategies include an engineered ‘vehicle’, which may be a porous scaffold or a dense substrate or patch, made of either natural or synthetic polymeric materials. The function of the substrate is to aid transportation of cells into the diseased region of the heart and support their integration. This book, which contains chapters written by leading experts in MTE, gives a complete analysis of the area and presents the latest advances in the field. The chapters cover all relevant aspects of MTE strategies, including cell sources, specific TE techniques and biomaterials used. Many different cell types have been suggested for cell therapy in the framework of MTE, including autologous bone marrow-derived or cardiac progenitors, as well as embryonic or induced pluripotent stem cells, each having their particular advantages and disadvantages. The book covers a complete range of biomaterials, examining different aspects of their application in MTE, such as biocompatibility with cardiac cells, mechanical capability and compatibility with the mechanical properties of the native myocardium as well as degradation behaviour in vivo and in vitro. Although a great deal of research is being carried out in the field, this book also addresses many questions that still remain unanswered and highlights those areas in which further research efforts are required. The book will also give an insight into clinical trials and possible novel cell sources for cell therapy in MTE.

By combining tissue engineering techniques with human-induced pluripotent stem cell (hiPSC) technology, human-derived engineered cardiac tissues (ECTs) have been developed using several cell lineage compositions and 3-dimensional geometries. Although hiPSC ECTs are relatively immature compared with native adult heart tissues, they have promising potential as a platform technology for drug-screening and disease modeling, and as grafts for hiPSC-based regenerative heart therapy. This chapter provides the focused overview of the current status of cardiac tissue engineering technology and its possible application.

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.

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: 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.