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.

Though the heart is one of the first organs to develop during embryogenesis and the physical aspects of development are well documented, little is known of the molecular mechanisms that control heart development. BMP signaling has been implicated in cardiac development both in vivo and in vitro, the initial research focused on altering this pathway. BMP signaling belongs to the signaling superfamily of transforming growth factor-b (Tgf-(beta)). Further evidence from mouse knockout studies, reveals a critical role of signaling through the Tgf-(beta) receptors in which Tgf-(beta) 3-/- mice demonstrate congenital heart defects. Tgf-(beta) signaling is typically relayed through a tetramer complex composed of two Tgf-(beta) type II and two type I (ALK5) receptors. The signaling of this tetramer has recently been identified in the differentiation of epicardial and endocardial to mesenchyme. Proceeding experiments have demonstrated that knocking ALK5 out selectively in endocardium, myocardium, or epicardium does not interfere with normal cardiac muscle development in vivo. Sridurongrit suggest that ALK5 signaling is required for smooth muscle development and vascularization of the myocardium but not cardiomyocoyte development. Therefore the role of ALK5 signaling during cardiac development is studied int two pluripotent models, mouse embryonic stem cells and human induced pluripotent stem cells (hiPS) in this research to understand the role of this pathway in cardiogenesis. Further the ultimate goals of this research is to screen small molecules and develop protocols that direct diffentiation of pluripotent stem cells to mesoderm and ultimately a cardiomyocyte fate. There are two major differentiation events that occur as a pluripotent stem cell differentiates to a terminal state. The cell begins as a pluripotent cell that can give rise to all somatic cell types as this cell differentiates it enters multipotent stage. Multipotent cells become partially programmed and can give rise to only certain somatic fates. These multipotent progenitors will ultimately give rise to structured tissue composed of specific somatic cell types. However, the molecular pathways that control differentiation to specific somatic fates remain poorly understood. The focus of this research is to explore these pathways using small molecule inhibitors to better understand the internal cell signaling that controls cardiogenesis. The research presented in this paper occurs in two major stages. First the experiments focus on developing protocols that can induce pluripotent stem cells to give rise to mesoderm, the germ layer from which cardiomyocytes are derived. Secondly, small molecules are screened to understand their ability to drive this mesoderm to a cardiomyocyte fate. Exploring these pathways, that control cardiogenesis, is essential if stem cells are to provide a supply of primary cardiomycotes to better understand human cardiac physiology and the affect potential drugs will have on their function. Heart disease remains the number one cause of death in the developed world. Therefore there is not only a need to develop novel molecules that can assuage cardiac disease but there is also a need to understand how these diseases develop. hiPS have the potential to fulfill both these needs. These cells can be derived directly from patients with specific cardiac afflictions. By controlling the differentiation of these disease derived pluripotent cells, researchers will be able to track physical and chemical changes in cardiomyocyte development that ultimately lead to a diseased phenotype. This creates a powerful tool to study new molecules and cardiac disease. Screening of small molecules that alter the diseased phenotype of these patients will further understanding of chemical modulation of cardiomyocytes and the ability of potential drugs to mitigate disease. This research has the potential to ultimately lead to patient specific therapeutics in the treatment of heart disease.

To achieve cardiac regeneration using pluripotent stem (iPS) cells, researchers must understand iPS cell generation methods, cardiomyocyte differentiation protocols, cardiomyocyte characterization methods, and tissue engineering. This book presents the current status and future possibilities in cardiac regeneration using iPS cells. Written by top researchers who present new data in these fields, this book reviews cardiac cell therapy for ischemic heart disease and explores in vitro generation of efficacious platelets from iPS cells. It also discusses modeling arrhythmogenic heart disease with patient-specific induced pluripotent stem cells.

Tissue engineering combined with the power of stem cell technology provides unprecedented opportunity to interrogate and discover human biology. The derivation of human cardiomyocytes from induced pluripotent stem cells (iPSCs) has enabled the development of de novo cardiac tissues that recapitulate several characteristics of functional human myocardium. These platforms act as advanced models with which cardiac development, biomechanics, and disease pathology can be reverse engineered to glean otherwise unobtainable information that has great significance to human health. Despite this progress, tissue engineering is in its infancy, and the ability to produce cardiac tissues that mimic the function of mature adult myocardium has proven to be quite challenging. Using immature tissues for disease modeling or developing novel therapeutics poses a potential danger for generating falsely positive or negative results. To combat this risk, there has been significant effort in developing biofabrication strategies to recapitulate the complex tissue architecture of the myocardium. However, there are few platforms available that can recreate the multiscale organization of the heart, including tissue anisotropy, helical myocardial organization, and ventricular and atrial chamber geometries. To address this shortcoming, we have developed a nanopatterned cell sheet technology for fabrication of complex 3D cardiac ventricular models with controllable cellular architecture. In this approach flexible thermoresponsive nanofabricated substrates (fTNFS) were used to create sheets of organized cardiac tissue and cast them into simplified chamber geometries such as a hollow tube or cone shape. These tissues exhibited spontaneous beating and could create measurable pumping pressures. Additionally, we measured functional benefits from tissues with anisotropic cellular patterning as compared to tissues with non-biomimetic cellular arrangements. We also observed that tissues patterned with circumferential cellular alignment exhibited surprising cellular remodeling where the cells rotated almost 90 degrees in orientation from their initial circumferential pattern. Upon modeling this effect computationally using finite element analysis, we discovered large gradients of shear stress perpendicular to cellular alignment that may have motivated cellular reorganization. Together, these findings demonstrate the importance of mimicking myocardial architecture in engineered tissue models. These works provide an advanced platform for studying tissue structure-function relationships in cardiac development and disease.

Angiogenesis, the development of new blood vessels from the existing vasculature, is essential for physiological growth and over 18,000 research articles have been published describing the role of angiogenesis in over 70 different diseases, including cancer, diabetic retinopathy, rheumatoid arthritis and psoriasis. One of the most important technical challenges in such studies has been finding suitable methods for assessing the effects of regulators of eh angiogenic response. While increasing numbers of angiogenesis assays are being described both in vitro and in vivo, it is often still necessary to use a combination of assays to identify the cellular and molecular events in angiogenesis and the full range of effects of a given test protein. Although the endothelial cell - its migration, proliferation, differentiation and structural rearrangement - is central to the angiogenic process, it is not the only cell type involved. the supporting cells, the extracellular matrix and the circulating blood with its cellular and humoral components also contribute. In this book, experts in the use of a diverse range of assays outline key components of these and give a critical appraisal of their strengths and weaknesses. Examples include assays for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures, assessment of endothelial and mural cell interactions, and such in vivo assays as the chick chorioallantoic membrane, zebrafish, corneal, chamber and tumour angiogenesis models. These are followed by a critical analysis of the biological end-points currently being used in clinical trials to assess the clinical efficacy of anti-angiogenic drugs, which leads into a discussion of the direction future studies should take. This valuable book is of interest to research scientists currently working on angiogenesis in both the academic community and in the biotechnology and pharmaceutical industries. Relevant disciplines include cell and molecular biology, oncology, cardiovascular research, biotechnology, pharmacology, pathology and physiology.

Advances in Tissue Engineering is a unique volume and the first of its kind to bring together leading names in the field of tissue engineering and stem cell research. A relatively young science, tissue engineering can be seen in both scientific and sociological contexts and successes in the field are now leading to clinical reality. This book attempts to define the path from basic science to practical application. A contribution from the UK Stem Cell Bank and opinions of venture capitalists offer a variety of viewpoints, and exciting new areas of stem cell biology are highlighted. With over fifty stellar contributors, this book presents the most up-to-date information in this very topical and exciting field./a

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.