Stem cell models of cardiac development and disease.

The past few years have witnessed remarkable advances in stem cell biology and human genetics, and we have arrived at an era in which patient-specific cell and tissue models are now practical. The recent identification of cardiovascular progenitor cells, as well as the identification of genetic variants underlying congenital heart disorders and adult disease, opens the door to the development of human models of human cardiovascular disease. We review the current understanding of the contribution of progenitor cells to cardiogenesis and outline how pluripotent stem cells can be applied to the modeling of cardiovascular disorders of genetic origin. A key challenge will be to implement these models in an efficient manner to develop a molecular understanding of how genes lead to disease and to screen for genes and drugs that modify the disease process.

Transient secretion of VEGF protein from transplanted hiPSC-CMs enhances engraftment and improves rat heart function post MI.

Cell-based therapies offer an exciting and novel treatment for heart repair following myocardial infarction (MI). However, these therapies often suffer from poor cell viability and engraftment rates, which involve many factors, including the hypoxic conditions of the infarct environment. Meanwhile, vascular endothelial growth factor (VEGF) has previously been employed as a therapeutic agent to limit myocardial damage and simultaneously induce neovascularization. This study took an approach to transiently overexpress VEGF protein, in a controlled manner, by transfecting human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with VEGF mRNA prior to transplantation. The conditioning of iPSC-CMs with VEGF mRNA ultimately led to greater survival rates of the transplanted cells, which promoted a stable vascular network in the grafted region. Furthermore, bulk RNA transcriptomics data and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) and AGE-RAGE signaling pathways were significantly upregulated in the VEGF-treated iPSC-CMs group. The over-expression of VEGF from iPSC-CMs stimulated cell proliferation and partially attenuated the hypoxic environment in the infarcted area, resulting in reduced ventricular remodeling. This study provides a valuable solution for the survival of transplanted cells in tissue-engineered heart regeneration and may further promote the application of modified mRNA (modRNA) in the field of tissue engineering.

Direct intramyocardial injection of VEGF mRNA in patients undergoing coronary artery bypass grafting.

Vascular endothelial growth factor A (VEGF-A) has therapeutic cardiovascular effects, but delivery challenges have impeded clinical development. We report the first clinical study of naked mRNA encoding VEGF-A (AZD8601) injected into the human heart. EPICCURE (ClinicalTrials.gov: NCT03370887) was a randomized, double-blind study of AZD8601 in patients with left ventricular ejection fraction (LVEF) 30%-50% who were undergoing elective coronary artery bypass surgery. Thirty epicardial injections of AZD8601 (total 3 mg) or placebo in citrate-buffered saline were targeted to ischemic but viable myocardial regions mapped using quantitative [15O]-water positron emission tomography. Seven patients received AZD8601 and four received placebo and were followed for 6 months. There were no deaths or treatment-related serious adverse events and no AZD8601-associated infections, immune reactions, or arrhythmias. Exploratory outcomes indicated potential improvement in LVEF, Kansas City Cardiomyopathy Questionnaire scores, and N-terminal pro-B-type natriuretic peptide levels, but the study is limited in size, and significant efficacy conclusions are not possible from the dataset. Naked mRNA without lipid encapsulation may provide a safe delivery platform for introducing genetic material to cardiac muscle, but further studies are needed to confirm efficacy and safety in a larger patient pool.

Origins and fates of cardiovascular progenitor cells.

Multipotent cardiac progenitor cells are found in the fetal and adult heart of many mammalian species including humans and form as intermediates during the differentiation of embryonic stem cells. Despite similar biological properties, the molecular identities of these different cardiac progenitor cell populations appear to be distinct. Elucidating the origins and lineage relationships of these cell populations will accelerate clinical applications such as drug screening and cell therapy as well as shedding light on the pathogenic mechanisms underlying cardiac diseases.

Cardiac progenitors and paracrine mediators in cardiogenesis and heart regeneration.

The mammalian hearts have the least regenerative capabilities among tissues and organs. As such, heart regeneration has been and continues to be the ultimate goal in the treatment against acquired and congenital heart diseases. Uncovering such a long-awaited therapy is still extremely challenging in the current settings. On the other hand, this desperate need for effective heart regeneration has developed various forms of modern biotechnologies in recent years. These involve the transplantation of pluripotent stem cell-derived cardiac progenitors or cardiomyocytes generated in vitro and novel biochemical molecules along with tissue engineering platforms. Such newly generated technologies and approaches have been shown to effectively proliferate cardiomyocytes and promote heart repair in the diseased settings, albeit mainly preclinically. These novel tools and medicines give somehow credence to breaking down the barriers associated with re-building heart muscle. However, in order to maximize efficacy and achieve better clinical outcomes through these cell-based and/or cell-free therapies, it is crucial to understand more deeply the developmental cellular hierarchies/paths and molecular mechanisms in normal or pathological cardiogenesis. Indeed, the morphogenetic process of mammalian cardiac development is highly complex and spatiotemporally regulated by various types of cardiac progenitors and their paracrine mediators. Here we discuss the most recent knowledge and findings in cardiac progenitor cell biology and the major cardiogenic paracrine mediators in the settings of cardiogenesis, congenital heart disease, and heart regeneration.

An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation.

BACKGROUND: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. METHODS: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. RESULTS: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. CONCLUSION: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage.

A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.

Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors.

Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.

SMAD4 Is Essential for Human Cardiac Mesodermal Precursor Cell Formation.

Understanding stage-specific molecular mechanisms of human cardiomyocyte (CM) progenitor formation and subsequent differentiation are critical to identify pathways that might lead to congenital cardiovascular defects and malformations. In particular, gene mutations in the transforming growth factor (TGF)ß superfamily signaling pathways can cause human congenital heart defects, and murine loss of function studies of a central component in this pathway, Smad4, leads to early embryonic lethality. To define the role of SMAD4 at the earliest stages of human cardiogenesis, we generated SMAD4 mutant human embryonic stem cells (hESCs). Herein, we show that the loss of SMAD4 has no effect on hESC self-renewal, or neuroectoderm formation, but is essential for the formation of cardiac mesoderm, with a subsequent complete loss of CM formation during human ES cell cardiogenesis. Via transcriptional profiling, we show that SMAD4 mutant cell lines fail to generate cardiac mesodermal precursors, clarifying a role of NODAL/SMAD4 signaling in cardiac mesodermal precursor formation via enhancing the expression of primitive streak genes. Since SMAD4 relative pathways have been linked to congenital malformations, it will become of interest to determine whether these may due, in part, to defective cell fate decision during cardiac mesodermal precursor formation. Stem Cells 2018 Stem Cells 2019;37:216-225.

Programming and reprogramming a human heart cell.

The latest discoveries and advanced knowledge in the fields of stem cell biology and developmental cardiology hold great promise for cardiac regenerative medicine, enabling researchers to design novel therapeutic tools and approaches to regenerate cardiac muscle for diseased hearts. However, progress in this arena has been hampered by a lack of reproducible and convincing evidence, which at best has yielded modest outcomes and is still far from clinical practice. To address current controversies and move cardiac regenerative therapeutics forward, it is crucial to gain a deeper understanding of the key cellular and molecular programs involved in human cardiogenesis and cardiac regeneration. In this review, we consider the fundamental principles that govern the "programming" and "reprogramming" of a human heart cell and discuss updated therapeutic strategies to regenerate a damaged heart.

Human ISL1+ Ventricular Progenitors Self-Assemble into an In Vivo Functional Heart Patch and Preserve Cardiac Function Post Infarction.

The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm × 3 mm × 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy.

Insulin-Like Growth Factor 1 Receptor-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

BACKGROUND: Epicardial adipose tissue volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate epicardial adipose tissue's formation and expansion are unclear. METHODS: We used a novel modified mRNA-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart. RESULTS: Using 2 independent lineage-tracing strategies in murine models, we show that cells originating from the Wt1+ mesothelial lineage, which includes epicardial cells, differentiate into epicardial adipose tissue after myocardial infarction. This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation in the context of exogenous, IGF1-modified mRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1 lineage cell differentiation into adipocytes after myocardial infarction. CONCLUSIONS: Our results establish IGF1R signaling as a key pathway that governs epicardial adipose tissue formation in the context of myocardial injury by redirecting the fate of Wt1+ lineage cells. Our study also demonstrates the power of modified mRNA -based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.

How to make a cardiomyocyte.

During development, cardiogenesis is orchestrated by a family of heart progenitors that build distinct regions of the heart. Each region contains diverse cell types that assemble to form the complex structures of the individual cardiac compartments. Cardiomyocytes are the main cell type found in the heart and ensure contraction of the chambers and efficient blood flow throughout the body. Injury to the cardiac muscle often leads to heart failure due to the loss of a large number of cardiomyocytes and its limited intrinsic capacity to regenerate the damaged tissue, making it one of the leading causes of morbidity and mortality worldwide. In this Primer we discuss how insights into the molecular and cellular framework underlying cardiac development can be used to guide the in vitro specification of cardiomyocytes, whether by directed differentiation of pluripotent stem cells or via direct lineage conversion. Additional strategies to generate cardiomyocytes in situ, such as reactivation of endogenous cardiac progenitors and induction of cardiomyocyte proliferation, will also be discussed.

Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages.

The generation and expansion of diverse cardiovascular cell lineages is a critical step during human cardiogenesis, with major implications for congenital heart disease. Unravelling the mechanisms for the diversification of human heart cell lineages has been hampered by the lack of genetic tools to purify early cardiac progenitors and define their developmental potential. Recent studies in the mouse embryo have identified a multipotent cardiac progenitor that contributes to all of the major cell types in the murine heart. In contrast to murine development, human cardiogenesis has a much longer onset of heart cell lineage diversification and expansion, suggesting divergent pathways. Here we identify a diverse set of human fetal ISL1(+) cardiovascular progenitors that give rise to the cardiomyocyte, smooth muscle and endothelial cell lineages. Using two independent transgenic and gene-targeting approaches in human embryonic stem cell lines, we show that purified ISL1(+) primordial progenitors are capable of self-renewal and expansion before differentiation into the three major cell types in the heart. These results lay the foundation for the generation of human model systems for cardiovascular disease and novel approaches for human regenerative cardiovascular medicine.

Regenerative medicine and human models of human disease.

Recent advances in stem-cell technology are now allowing the mechanisms of human disease to be studied in human cells. A new era for regenerative medicine is arising from such disease models, extending beyond early cell-based therapies and towards evaluating genetic variation in humans and identifying the molecular pathways that lead to disease, as well as targets for therapy.

Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart.

The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

Derivation of human primordial germ cell-like cells in an embryonic-like culture.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.