Human embryonic and induced pluripotent stem cells (collectively known as pluripotent stem cells or hPSCs) can be a renewable source of cardiomyocytes for treating heart diseases which are leading causes of morbidity and mortality. Recent advances in the generation of patient-specific induced PSCs greatly improve the chances of clinical success. Yet, robust methods are still under development for producing specific cardiac muscle cell-types across different hPSC lines having varying lineage-specific differentiation propensities. This requires a detailed understanding of signaling pathways and differentiation mechanisms in context of several ambient conditions such as cell epigenetic profile, endogenous signaling, and cell differentiation state. Therapeutic realization also hinges on the transplantation of sufficient numbers of cells (approximately 1. 5x109 per damaged myocardium [1]) pointing to the need for pertinent large-scale bioprocesses. In this study a novel, hormone-free, xeno-free medium is developed that is devoid of signaling activity. Since contemporary xeno-free formulations contain agents with signaling activity (such as growth hormone, retinoic acid, corticosterone, LiCl, ascorbic acid) that are reported to interfere with cardiac differentiation, proper assessment of signaling mechanisms is not feasible. Therefore a factorial design consisting of 73 conditions is employed a develop a medium comprising of specific combination of basal medium, iron-carrier (holo-transferrin), reducing agent (selenium), and a novel cocktail to increase cell growth and viability (termed XFM cocktail) containing optimized levels of lipids, aminoacids and vitamins. The medium developed here is utilized to investigate signaling mechanisms governing cardiomyocyte differentiation of hPSCs. The simple formulation of the medium offers significant economic advantages over other xeno-free media for a large scale differentiation process. Finally, since the medium is free from xenogeneic and allogeneic agents (such as albumin), clinical translation is feasible preventing the risk of transferring animal-derived pathogens and non-native proteins that can trigger adverse foreign-body immune reactions and possible implant rejection. This is the first report of accurate assessment of majority of physiologically-relevant signaling pathways elucidating their individual roles in cardiac differentiation as well as formation of closely-related lineages. By employing a design-of-experiment approach, an efficient path of hPSC-to-cardiomyocyte differentiation is discovered comprising of two steps. A synergistic combination of BMP and Wnt3A treatment with endogenous FGF and Nodal signaling is determined for obtaining an intermediate progenitor-like stage termed mesoderm-oriented primitive streak (MePS) that significantly increases differentiation efficiency. Adjustment of treatment concentration and duration during the first step and appropriate time of starting the second step allows for efficient conversion of MePS cells to cardiomyoctes in a total of 6 days of differentiation. This optimization renders the method highly reproducible that translates across the most diverse set of hPSC lines with varied lineage-specific differentiation propensities. The efficiency of hPSC-to-cardiomyocyte conversion is >90% and the cell yield is the highest reported yet in the literature providing 23 cardiomyocytes per stem cell seeded or 0. 9 million cardiomyocytes/cm2. First beating is seen as early as day 6 of differentiation and by day 8 there is interconnected and synchronized beating activity with majority of cells beating in each dish. By day 16 of differentiation, formation of bi-nucleated cells is observed indicating maturation toward adult-like cardiomyocytes. Distribution of cardiac cell-types is determined by electrophysiological measurements to be ~60% ventricular and ~40% atrial and sinoatrial node. Investigation of molecular mechanisms in the first stage reveals a crosstalk between BMP and Wnt pathways providing novel scientific information regarding mechanisms governing efficient cardiomyocyte differentiation. In the next stage, pathways are elucidated that selectively pattern primitive streak cells to different mesoderm derivatives such as smooth muscle, hematopoietic, blood and cardiac. Initial results demonstrate increased cardiac gene expression and modulation of atrial and ventricular markers providing avenues for fine-tuning mesoderm into different cardiac cell-types and possibly for increasing yield of differentiation. The outcome of this thesis is expected to facilitate the development of stem cell-based therapies for the heart.

The heart can be considered the most important organ of our body, as it supplies nutrients to all the cells. When affected from injuries or diseases, the heart function is hampered, as the damaged area is substituted by a fibrotic scar instead of functional tissue. Understanding the mechanisms leading to heart failure and finding a cure for cardiac diseases represents a major challenge of modern medicine, since they are the leading cause of death and disability in Western world. Being the heart a vital organ it is difficult to have access to its cells, especially in humans. In order to model it or find therapeutic strategies many approaches and cell sources have been studied. For example cardiac stem cells, skeletal myoblasts, bone marrow-derived cells and peripheral blood mononuclear cells have been tested in pre-clinical and clinical trials, without significant tissue regeneration. Human pluripotent stem cells (hPSC) are thought to be the most promising cell type in the field, thanks to their unlimited capacity of self-renewal and retention of differentiation potency. Induced pluripotent stem cells (iPSC) are pluripotent cells derived through reprogramming from adult cells, easily accessible from patients, like keratinocytes. iPSC can be differentiated to cardiac cells, through stage-specific protocols that reproduce embryonic development, offering a very useful platform for modelling diseases of patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, properly mimicking cardiac tissue is very complex, since not only the correct cardiac cell type has to be reproduced, but also its overall cellular composition, architecture and biophysical functions. In order to study these aspects, we applied biotechnological strategies such as the use of transgenic cell lines for obtaining pure and scalable differentiated cells to be cultured in a 3D scaffold with a perfusion bioreactor. Although it is well known that iPSC can give rise to cardiomyocytes in vitro, not every cell line can be efficiently differentiated. Thus, a cell line-specific differentiation protocol has to be identified and optimized. We finally identified a fast and efficient stage-specific differentiation protocol suitable for the iPSC lines used in this work, derived from human keratinocytes. With this protocol, we can reproducibly obtain close to 50% cardiomyocytes after 15 days of differentiation. One important feature of currently available differentiation protocols is that the target cell type is obtained among a heterogeneous cell population. To track the cardiac population of interest we generated transgenic cell lines where the reporter protein GFP follows the expression of different genes specific for stages of differentiation, such as T (Brachyury) for mesoderm; NKX2.5 for cardiac progenitors; and MHC for cardiomyocytes. Moreover, cardiomyocytes obtained from hPSC using currently available differentiation protocols are typically immature, mostly resembling embryonic or fetal cardiomyocytes, arguably because of the lack of mechanical and electrical stimuli that only a 3D environment can provide. In order to create a piece of tissue in 3D we used a collagen and elastin-based scaffold, to mimic the structural proteins of endogenous extracellular matrix. We also built a perfusion bioreactor to culture the construct. After initial validation with primary cultures of rat neonatal cardiomyocytes, we tested iPSC-derived cardiac cells at different stages of differentiation. While early mesoderm or cardiac progenitors could not survive in our system, iPSC differentiated to cardiomyocytes, could be retained and maintained alive within the scaffold for at least 4 days. In conclusion, in this work we combined biotechnological tools in order to obtain a test platform for studying the mechanisms underlying cardiac differentiation, maturation, as well as providing valuable in vitro systems for disease modelling, drug screening of patient-specific heart muscle cells and cell therapy.

The book covers multifarious aspects of stem cell-based therapy for cardiovascular diseases. In addition to stem cells from different sources for cell-based therapy, it covers stem cell organoids and stem cell-derived exosomes in regenerative medicine. The book also encompasses advances in state-of-the-art infrastructure to improve the maturation aspects of pluripotent stem cells-derived cardiomyocytes using a novel scaffold-based cell culture system for cell delivery in experimental animal models and clinical settings. Besides the use of mesenchymal stem cells, the book includes chapters on the use of cardiac progenitor cells (CPCs), microtissue implantation, use of PSCs for valvulopathies, application of de-cellularized organ arrays as natural scaffolds for cardiac tissue engineering, use of epicardial stem cells, and skeletal myoblasts in cell-based therapy for myocardial regeneration. Besides the cell-based therapy approach, the book also reviews the stem cell-derived exosomes, their characteristics, and engineering strategies to enhance their therapeutic potential via targeting and drug loading and use in disease models. Additionally, the book also discusses the latest research on injectable hydrogels for cardiovascular regeneration and how hydrogel-based delivery protects the cells and their retention post-engraftment in the heart, a problem, which significantly reduces the efficacy of cell-based therapy.

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.

There is hardly an area of research developing so quickly and raising so many promises as stem cell research. Adult, embryonic and recently available induced pluripotent stem cells not only foster our understanding of differentiation of endo-, ecto- and mesodermal lineages to all organs of the body, but foremost nourish the hope that cells grown in culture can be used for regeneration of diseased organs such as the heart damaged by myocardial infarction. This book focuses on perspectives of stem cells for regenerative therapy of cardiovascular diseases. Based on the EC consortium INELPY, it reviews the field and disseminates major outcomes of this project. Thus it introduces the reader to this fascinating area of research and incorporates very recent findings interesting to the expert, spanning the field from bench to bedside. The compilation of contributions is unique as there is yet no similar comprehensive overview combining stem cell research with preclinical and clinical evaluation as well as engineering of tissue patches for transplantation. As such it will be an invaluable source of information for all researchers in the stem cell and tissue regeneration field including bioengineers as well as for all clinicians interested in regenerative therapies, especially for ischemic cardiomyopathies.

In excess of 7 million people worldwide die of coronary heart disease each year. Only one-third of these heart attack victims recover completely. The remainder suffer the consequences of myocardial infarction and its ill fated remodeling process, resulting in chronic congestive heart failure. This malady alone is the leading cause of hospital admissions in the United States. New breakthroughs in stem cell therapy and tissue engineering have promised to reverse this dismal outcome by cardiovascular repair. World authorities, including scientists and regulatory authorities, have joined in a collaborative effort to present for the reader the first collective review of stem cell therapy for the treatment of cardiovascular disease. These contributions in basic science, pre-clinical and clinical experience guided by the regulatory pathways, assure a rapid course of translational research and clinical trials. The contents of this publication will become a prerequisite for those preparing to meet the challenges of this exciting and potentially rewarding field of stem cell research.

Stem Cell and Gene Therapy for Cardiovascular Disease is a state-of-the-art reference that combines, in one place, the breadth and depth of information available on the topic. As stem cell and gene therapies are the most cutting-edge therapies currently available for patients with heart failure, each section of the book provides information on medical trials from contributors and specialists from around the world, including not only what has been completed, but also what is planned for future research and trials. Cardiology researchers, basic science clinicians, fellows, residents, students, and industry professionals will find this book an invaluable resource for further study on the topic. - Provides information on stem and gene therapy medical trials from contributors and specialists around the world, including not only what has been completed, but also what is planned for future research and trials - Presents topics that can be applied to allogeneic cells, mesenchymal cells, gene therapy, cardiomyoctyes, iPS cells, MAPC's, and organogenesis - Covers the three areas with the greatest clinical trials to date: chronic limb ischemia, chronic angina, and acute MI - Covers the prevailing opinions on how to harness the body's natural repair mechanisms - Ideal resource for cardiology researchers, basic science clinicians, fellows, residents, students, and industry professionals