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.

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.

There is great potential for human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) to serve as a test bed for developmental, pharmacological, and regenerative studies. These cells can serve as therapeutic agents, which can be implanted into damage heart tissue to supplant dead cells. They can be used to assess new pharmacological treatments for heart disease. Moreover, they can be used as model systems to study the progression of developmental and pathological states of the heart. However, upon differentiation into cardiomyocytes, these cells are distinctly immature i.e. their cell size, shape, cardiac-specific markers, ploidy, nucleation, calcium handling properties, action potentials, contractility, metabolism, etc. more closely mimic that of an embryonic-stage cardiomyocyte. Therefore, in order for these cells to serve as a valid replacement or model for more developed cardiomyocytes, their structural and functional maturation must be assessed and enhanced. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. Therefore, having the ability to quantify this contraction would provide a powerful assessment tool for hPSC-CMs. Arrays of micropost have previously been employed as a means to measure the isotonic contraction of cardiomyocytes. In this work, a new micropost technique was developed in order to allow for real-time measurements of hPSC-CM contractility, to enable contractile assessment under various different culture conditions. Previous studies with immature cardiomyocytes have shown that a number of different methods are able to enhance their contractile and structural maturation. Here, hPSC-CM maturation was achieved via: i) prolonged cell culture, ii) cell alignment, iii) controlling cell-cell contact between adjacent cells, iv) altering substrate stiffness, v) electrically-stimulating the cells, and vii) treating the cells with various different biochemical agents. Assessment of hPSC-CM structural maturation was achieved by immunofluorescent analysis, while high speed imaging of micropost deflections and fluorescent calcium transients was used to quantify functional maturation. Through these studies, I found that the micropost assay is capable of accessing the contractile state of immature human cardiomyocytes, which makes it a powerful tool for developmental studies, pharmacological screening, and disease modeling applications. Furthermore, the pro-maturation environment that I developed was able to elicit cardiomyocyte maturation in the absence of any biochemical cues. Ultimately, I believe that these novel culture and analysis techniques will provide future researchers with a means to culture large populations of rapidly matured stem cell-derived cardiomyocytes, in order to effectively perform developmental, pharmacological, and therapeutic studies in a more rapid and high-throughput manner.

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.

A comprehensive overview of the latest achievements, trends, and the current state of the art of this important and rapidly expanding field. Clearly and logically structured, the first part of the book explores the fundamentals of tissue engineering, providing a separate chapter on each of the basic topics, including biomaterials stem cells, biosensors and bioreactors. The second part then follows a more applied approach, discussing various applications of tissue engineering, such as the replacement or repairing of skins, cartilages, livers and blood vessels, to trachea, lungs and cardiac tissues, to musculoskeletal tissue engineering used for bones and ligaments as well as pancreas, kidney and neural tissue engineering for the brain. The book concludes with a look at future technological advances. An invaluable reading for entrants to the field in biomedical engineering as well as expert researchers and developers in industry.

This open access book focuses on the molecular mechanism of congenital heart disease and pulmonary hypertension, offering new insights into the development of pulmonary circulation and the ductus arteriosus. It describes in detail the molecular mechanisms involved in the development and morphogenesis of the heart, lungs and ductus arteriosus, covering a range of topics such as gene functions, growth factors, transcription factors and cellular interactions, as well as stem cell engineering technologies. The book also presents recent advances in our understanding of the molecular mechanism of lung development, pulmonary hypertension and molecular regulation of the ductus arteriosus. As such, it is an ideal resource for physicians, scientists and investigators interested in the latest findings on the origins of congenital heart disease and potential future therapies involving pulmonary circulation/hypertension and the ductus arteriosus.