Multi-chamber cardioids unravel human heart development and cardiac defects.

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

Maturing differentiated human pluripotent stem cells in vitro: methods and challenges.

Human pluripotent stem cells (hPSCs), derived from individuals or genetically modified with disease-related mutations and variants, have revolutionised studies of human disease. Researchers are beginning to exploit the extraordinary potential of stem cell technology to screen for new drugs to treat intractable diseases, ideally without side-effects. However, a major problem is that the differentiated cell types on which these models are based are immature; they resemble fetal and not adult cells. Here, we discuss the nature and hurdles of hPSC maturation, using cardiomyocytes as an example. We review methods used to induce cardiomyocyte maturation in culture and consider remaining challenges for their integration into research on human disease and drug development pipelines.

In vitro models of the human heart.

Cardiac congenital disabilities are the most common organ malformations, but we still do not understand how they arise in the human embryo. Moreover, although cardiovascular disease is the most common cause of death globally, the development of new therapies is lagging compared with other fields. One major bottleneck hindering progress is the lack of self-organizing human cardiac models that recapitulate key aspects of human heart development, physiology and disease. Current in vitro cardiac three-dimensional systems are either engineered constructs or spherical aggregates of cardiomyocytes and other cell types. Although tissue engineering enables the modeling of some electro-mechanical properties, it falls short of mimicking heart development, morphogenetic defects and many clinically relevant aspects of cardiomyopathies. Here, we review different approaches and recent efforts to overcome these challenges in the field using a new generation of self-organizing embryonic and cardiac organoids.

Three-dimensional cardiac models: a pre-clinical testing platform.

Major advancements in human pluripotent stem cell (hPSC) technology over recent years have yielded valuable tools for cardiovascular research. Multi-cell type 3-dimensional (3D) cardiac models in particular, are providing complementary approaches to animal studies that are better representatives than simple 2-dimensional (2D) cultures of differentiated hPSCs. These human 3D cardiac models can be broadly divided into two categories; namely those generated through aggregating pre-differentiated cells and those that form self-organizing structures during their in vitro differentiation from hPSCs. These models can either replicate aspects of cardiac development or enable the examination of interactions among constituent cell types, with some of these models showing increased maturity compared with 2D systems. Both groups have already emerged as physiologically relevant pre-clinical platforms for studying heart disease mechanisms, exhibiting key functional attributes of the human heart. In this review, we describe the different cardiac organoid models derived from hPSCs, their generation methods, applications in cardiovascular disease research and use in drug screening. We also address their current limitations and challenges as pre-clinical testing platforms and propose potential improvements to enhance their efficacy in cardiac drug discovery.

Of form and function: Early cardiac morphogenesis across classical and emerging model systems.

The heart is the earliest organ to develop during embryogenesis and is remarkable in its ability to function efficiently as it is being sculpted. Cardiac heart defects account for a high burden of childhood developmental disorders with many remaining poorly understood mechanistically. Decades of work across a multitude of model organisms has informed our understanding of early cardiac differentiation and morphogenesis and has simultaneously opened new and unanswered questions. Here we have synthesized current knowledge in the field and reviewed recent developments in the realm of imaging, bioengineering and genetic technology and ex vivo cardiac modeling that may be deployed to generate more holistic models of early cardiac morphogenesis, and by extension, new platforms to study congenital heart defects.

Recent advances in pluripotent stem cell-derived cardiac organoids and heart-on-chip applications for studying anti-cancer drug-induced cardiotoxicity.

Cardiovascular disease (CVD) caused by anti-cancer drug-induced cardiotoxicity is now the second leading cause of mortality among cancer survivors. It is necessary to establish efficient in vitro models for early predicting the potential cardiotoxicity of anti-cancer drugs, as well as for screening drugs that would alleviate cardiotoxicity during and post treatment. Human induced pluripotent stem cells (hiPSCs) have opened up new avenues in cardio-oncology. With the breakthrough of tissue engineering technology, a variety of hiPSC-derived cardiac microtissues or organoids have been recently reported, which have shown enormous potential in studying cardiotoxicity. Moreover, using hiPSC-derived heart-on-chip for studying cardiotoxicity has provided novel insights into the underlying mechanisms. Herein, we summarize different types of anti-cancer drug-induced cardiotoxicities and present an extensive overview on the applications of hiPSC-derived cardiac microtissues, cardiac organoids, and heart-on-chips in cardiotoxicity. Finally, we highlight clinical and translational challenges around hiPSC-derived cardiac microtissues/organoids/heart-on chips and their applications in anti-cancer drug-induced cardiotoxicity. • Anti-cancer drug-induced cardiotoxicities represent pressing challenges for cancer treatments, and cardiovascular disease is the second leading cause of mortality among cancer survivors. • Newly reported in vitro models such as hiPSC-derived cardiac microtissues/organoids/chips show enormous potential for studying cardio-oncology. • Emerging evidence supports that hiPSC-derived cardiac organoids and heart-on-chip are promising in vitro platforms for predicting and minimizing anti-cancer drug-induced cardiotoxicity.

Advances in 3D Organoid Models for Stem Cell-Based Cardiac Regeneration.

The adult human heart cannot regain complete cardiac function following tissue injury, making cardiac regeneration a current clinical unmet need. There are a number of clinical procedures aimed at reducing ischemic damage following injury; however, it has not yet been possible to stimulate adult cardiomyocytes to recover and proliferate. The emergence of pluripotent stem cell technologies and 3D culture systems has revolutionized the field. Specifically, 3D culture systems have enhanced precision medicine through obtaining a more accurate human microenvironmental condition to model disease and/or drug interactions in vitro. In this study, we cover current advances and limitations in stem cell-based cardiac regenerative medicine. Specifically, we discuss the clinical implementation and limitations of stem cell-based technologies and ongoing clinical trials. We then address the advent of 3D culture systems to produce cardiac organoids that may better represent the human heart microenvironment for disease modeling and genetic screening. Finally, we delve into the insights gained from cardiac organoids in relation to cardiac regeneration and further discuss the implications for clinical translation.

An evidence appraisal of heart organoids in a dish and commensurability to human heart development in vivo.

Stem-cell derived in vitro cardiac models have provided profound insights into mechanisms in cardiac development and disease. Efficient differentiation of specific cardiac cell types from human pluripotent stem cells using a three-step Wnt signaling modulation has been one of the major discoveries that has enabled personalized cardiovascular disease modeling approaches. Generation of cardiac cell types follow key development stages during embryogenesis, they intuitively are excellent models to study cardiac tissue patterning in primitive cardiac structures. Here, we provide a brief overview of protocols that have laid the foundation for derivation of stem-cell derived three-dimensional cardiac models. Further this article highlights features and utility of the models to distinguish the advantages and trade-offs in modeling embryonic development and disease processes. Finally, we discuss the challenges in improving robustness in the current models and utilizing developmental principles to bring higher physiological relevance. In vitro human cardiac models are complimentary tools that allow mechanistic interrogation in a reductionist way. The unique advantage of utilizing patient specific stem cells and continued improvements in generating reliable organoid mimics of the heart will boost predictive power of these tools in basic and translational research.

Reconstructing the heart using iPSCs: Engineering strategies and applications.

Induced pluripotent stem cells (iPSCs) have emerged as a key component of cardiac tissue engineering, enabling studies of cardiovascular disease mechanisms, drug responses, and developmental processes in human 3D tissue models assembled from isogenic cells. Since the very first engineered heart tissues were introduced more than two decades ago, a wide array of iPSC-derived cardiac spheroids, organoids, and heart-on-a-chip models have been developed incorporating the latest available technologies and materials. In this review, we will first outline the fundamental biological building blocks required to form a functional unit of cardiac muscle, including iPSC-derived cells differentiated by soluble factors (e.g., small molecules), extracellular matrix scaffolds, and exogenous biophysical maturation cues. We will then summarize the different fabrication approaches and strategies employed to reconstruct the heart in vitro at varying scales and geometries. Finally, we will discuss how these platforms, with continued improvements in scalability and tissue maturity, can contribute to both basic cardiovascular research and clinical applications in the future.

Multicellular Human Cardiac Organoids Transcriptomically Model Distinct Tissue-Level Features of Adult Myocardium.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.

Development of a human cardiac organoid injury model reveals innate regenerative potential.

The adult human heart possesses a limited regenerative potential following an ischemic event, and undergoes a number of pathological changes in response to injury. Although cardiac regeneration has been documented in zebrafish and neonatal mouse hearts, it is currently unknown whether the immature human heart is capable of undergoing complete regeneration. Combined progress in pluripotent stem cell differentiation and tissue engineering has facilitated the development of human cardiac organoids (hCOs), which resemble fetal heart tissue and can be used to address this important knowledge gap. This study aimed to characterize the regenerative capacity of immature human heart tissue in response to injury. Following cryoinjury with a dry ice probe, hCOs exhibited an endogenous regenerative response with full functional recovery 2 weeks after acute injury. Cardiac functional recovery occurred in the absence of pathological fibrosis or cardiomyocyte hypertrophy. Consistent with regenerative organisms and neonatal human hearts, there was a high basal level of cardiomyocyte proliferation, which may be responsible for the regenerative capacity of the hCOs. This study suggests that immature human heart tissue has an intrinsic capacity to regenerate.

Three-dimensional structural and metric characterisation of cardioids.

Exact three-dimensional (3D) structural information of developing organoids is key for optimising organoid generation and for studying experimental outcomes in organoid models. We set up a 3D imaging technique and studied complexly arranged native and experimentally challenged cardioids of two stages of remodelling. The imaging technique we employed is S-HREM (Scanning High Resolution Episcopic Microscopy), a variant of HREM, which captures multiple images of subsequently exposed surfaces of resin blocks and automatically combines them to large sized digital volume data of voxels sizes below 1 µm3. We provide precise volumetric information of the examined specimens and their single components and comparisons between stages in terms of volume and micro- and macroanatomic structure. We describe the 3D arrangement and lining of different types of cavities and their changes between day 10 and day 14 and map the various cell types to their precise spatial and structural environment. Exemplarily, we conducted semiautomatic counts of nuclei. In cryo-injured cardioids, we examined the extension and composition of the injured areas. Our results demonstrate the high quality and the great potential of digital volume data produced with S-HREM. It also provides sound metric and structural information, which assists production of native and experimentally challenged left ventricle cardioids and interpretation of their structural remodelling.

Improving human cardiac organoid design using transcriptomics.

Cardiovascular disease (CVD) is the leading cause of death worldwide. To this end, human cardiac organoids (hCOs) have been developed for improved organotypic CVD modeling over conventional in vivo animal models. Utilizing human cells, hCOs hold great promise to bridge key gaps in CVD research pertaining to human-specific conditions. hCOs are multicellular 3D models which resemble heart structure and function. Varying hCOs fabrication techniques leads to functional and phenotypic differences. To investigate heterogeneity across hCO platforms, we performed a transcriptomic analysis utilizing bulk RNA-sequencing from four previously published unique hCO studies. We further compared selected hCOs to 2D and 3D hiPSC-derived cardiomyocytes (hiPSC-CMs), as well as fetal and adult human myocardium bulk RNA-sequencing samples. Upon investigation utilizing Principal Component Analysis, K-means clustering analysis of key genes, and further downstream analyses such as Gene Set Enrichment (GSEA), Gene Set Variation (GSVA), and GO term enrichment, we found that hCO fabrication method influences maturity and cellular heterogeneity across models. Thus, we propose that adjustment of fabrication method will result in an hCO with a defined maturity and transcriptomic profile to facilitate its specified applications, in turn maximizing its modeling potential.

Cardiac construction-Recent advances in morphological and transcriptional modeling of early heart development.

During human embryonic development the early establishment of a functional heart is vital to support the growing fetus. However, forming the embryonic heart is an extremely complex process, requiring spatiotemporally controlled cell specification and differentiation, tissue organization, and coordination of cardiac function. These complexities, in concert with the early and rapid development of the embryonic heart, mean that understanding the intricate interplay between these processes that help shape the early heart remains highly challenging. In this review I focus on recent insights from animal models that have shed new light on the earliest stages of heart development. This includes specification and organization of cardiac progenitors, cell and tissue movements that make and shape the early heart tube, and the initiation of the first beat in the developing heart. In addition I highlight relevant in vitro models that could support translation of findings from animal models to human heart development. Finally I discuss challenges that are being addressed in the field, along with future considerations that together may help move us towards a deeper understanding of how our hearts are made.

Real-time electro-mechanical profiling of dynamically beating human cardiac organoids by coupling resistive skins with microelectrode arrays.

Cardiac organoids differentiated from induced pluripotent stem cells are emerging as a promising platform for pre-clinical drug screening, assessing cardiotoxicity, and disease modelling. However, it is challenging to simultaneously measure mechanical contractile forces and electrophysiological signals of cardiac organoids in real-time and in-situ with the existing methods. Here, we present a biting-inspired sensory system based on a resistive skin sensor and a microelectrode array. The bite-like contact can be established with a micromanipulator to precisely position the resistive skin sensor on the top of the cardiac organoid while the 3D microneedle electrode array probes from underneath. Such reliable contact is key to achieving simultaneous electro-mechanical measurements. We demonstrate the use of our system for modelling cardiotoxicity with the anti-cancer drug doxorubicin. The electro-mechanical parameters described here elucidate the acute cardiotoxic effects induced by doxorubicin. This integrated electro-mechanical system enables a suite of new diagnostic options for assessing cardiac organoids and tissues.

Guidelines for Manufacturing and Application of Organoids: Heart.

Cardiac organoids have emerged as invaluable tools for assessing the impact of diverse substances on heart function. This report introduces guidelines for general requirements for manufacturing cardiac organoids and conducting cardiac organoid-based assays, encompassing protocols, analytical methodologies, and ethical considerations. In the quest to employ recently developed three-dimensional cardiac organoid models as substitutes for animal testing, it becomes imperative to establish robust criteria for evaluating organoid quality and conducting toxicity assessments. This guideline addresses this need, catering to regulatory requirements, and describes common standards for organoid quality and toxicity assessment methodologies, commensurate with current technological capabilities. While acknowledging the dynamic nature of technological progress and the potential for future comparative studies, this guideline serves as a foundational framework. It offers a comprehensive approach to standardized cardiac organoid testing, ensuring scientific rigor, reproducibility, and ethical integrity in investigations of cardiotoxicity, particularly through the utilization of human pluripotent stem cell-derived cardiac organoids.

Cell cycle visualization tools to study cardiomyocyte proliferation in real-time.

Cardiomyocytes in the adult human heart are quiescent and those lost following heart injury are not replaced by proliferating survivors. Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.

Generative AI for Cell Type-Specific Fluorescence Image Generation of hPSC-derived Cardiac Organoid.

Human pluripotent stem cell (hPSC)-derived cardiac organoid is the most recent three-dimensional tissue structure that mimics the structure and functionality of the human heart and plays a pivotal role in modeling heart development and disease. The hPSC-derived cardiac organoids are commonly characterized by bright-field microscopic imaging for tracking daily organoid differentiation and morphology formation. Although the brightfield microscope provides essential information about hPSC-derived cardiac organoids, such as morphology, size, and general structure, it does not extend our understanding of cardiac organoids on cell type-specific distribution and structure. Then, fluorescence microscopic imaging is required to identify the specific cardiovascular cell types in the hPSC-derived cardiac organoids by fluorescence immunostaining fixed organoid samples or fluorescence reporter imaging of live organoids. Both approaches require extra steps of experiments and techniques and do not provide general information on hPSC-derived cardiac organoids from different batches of differentiation and characterization, which limits the biomedical applications of hPSC-derived cardiac organoids. This research addresses this limitation by proposing a comprehensive workflow for colorizing phase contrast images of cardiac organoids from brightfield microscopic imaging using conditional Generative Adversarial Networks (GANs) to provide cardiovascular cell type-specific information in hPSC-derived cardiac organoids. By infusing these phase contrast images with accurate fluorescence colorization, our approach aims to unlock the hidden wealth of cell type, structure, and further quantifications of fluorescence intensity and area, for better characterizing hPSC-derived cardiac organoids.

The new era of cardiovascular research: revolutionizing cardiovascular research with 3D models in a dish.

Cardiovascular research has heavily relied on studies using patient samples and animal models. However, patient studies often miss the data from the crucial early stage of cardiovascular diseases, as obtaining primary tissues at this stage is impracticable. Transgenic animal models can offer some insights into disease mechanisms, although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression. In recent years, a promising breakthrough has emerged in the form of in vitro three-dimensional (3D) cardiovascular models utilizing human pluripotent stem cells. These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment. This advancement is pivotal as it addresses the existing gaps in cardiovascular research, allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models. In this review, we first outline various approaches employed to generate these models. We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions. Moreover, we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety. Despite their immense potential, challenges persist, particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs. However, overcoming these challenges could revolutionize cardiovascular research. It has the potential to offer comprehensive mechanistic insights into human-specific disease processes, ultimately expediting the development of personalized therapies.

Design optimization of geometrically confined cardiac organoids enabled by machine learning techniques.

Stem cell organoids are powerful models for studying organ development, disease modeling, drug screening, and regenerative medicine applications. The convergence of organoid technology, tissue engineering, and artificial intelligence (AI) could potentially enhance our understanding of the design principles for organoid engineering. In this study, we utilized micropatterning techniques to create a designer library of 230 cardiac organoids with 7 geometric designs. We employed manifold learning techniques to analyze single organoid heterogeneity based on 10 physiological parameters. We clustered and refined the cardiac organoids based on their functional similarity using unsupervised machine learning approaches, thus elucidating unique functionalities associated with geometric designs. We also highlighted the critical role of calcium transient rising time in distinguishing organoids based on geometric patterns and clustering results. This integration of organoid engineering and machine learning enhances our understanding of structure-function relationships in cardiac organoids, paving the way for more controlled and optimized organoid design.