Upscaling Osteoclast Generation by Enhancing Macrophage Aggregation Using Hollow Microgels.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.

Metabolic Cardiomyopathies and Cardiac Defects in Inherited Disorders of Carbohydrate Metabolism: A Systematic Review.

Heart failure (HF) is a progressive chronic disease that remains a primary cause of death worldwide, affecting over 64 million patients. HF can be caused by cardiomyopathies and congenital cardiac defects with monogenic etiology. The number of genes and monogenic disorders linked to development of cardiac defects is constantly growing and includes inherited metabolic disorders (IMDs). Several IMDs affecting various metabolic pathways have been reported presenting cardiomyopathies and cardiac defects. Considering the pivotal role of sugar metabolism in cardiac tissue, including energy production, nucleic acid synthesis and glycosylation, it is not surprising that an increasing number of IMDs linked to carbohydrate metabolism are described with cardiac manifestations. In this systematic review, we offer a comprehensive overview of IMDs linked to carbohydrate metabolism presenting that present with cardiomyopathies, arrhythmogenic disorders and/or structural cardiac defects. We identified 58 IMDs presenting with cardiac complications: 3 defects of sugar/sugar-linked transporters (GLUT3, GLUT10, THTR1); 2 disorders of the pentose phosphate pathway (G6PDH, TALDO); 9 diseases of glycogen metabolism (GAA, GBE1, GDE, GYG1, GYS1, LAMP2, RBCK1, PRKAG2, G6PT1); 29 congenital disorders of glycosylation (ALG3, ALG6, ALG9, ALG12, ATP6V1A, ATP6V1E1, B3GALTL, B3GAT3, COG1, COG7, DOLK, DPM3, FKRP, FKTN, GMPPB, MPDU1, NPL, PGM1, PIGA, PIGL, PIGN, PIGO, PIGT, PIGV, PMM2, POMT1, POMT2, SRD5A3, XYLT2); 15 carbohydrate-linked lysosomal storage diseases (CTSA, GBA1, GLA, GLB1, HEXB, IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, ARSB, GUSB, ARSK). With this systematic review we aim to raise awareness about the cardiac presentations in carbohydrate-linked IMDs and draw attention to carbohydrate-linked pathogenic mechanisms that may underlie cardiac complications.

Highly parallelized human embryonic stem cell differentiation to cardiac mesoderm in nanoliter chambers on a microfluidic chip.

Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery. The generation, culture and differentiation of stem cells in low-volume, automated and parallelized microfluidic chips hold great promise to accelerate the research in this domain. Here, we show that we can differentiate human embryonic stem cells (hESCs) to early cardiac mesodermal cells in microfluidic chambers that have a volume of only 30 nanoliters, using discontinuous medium perfusion. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. During differentiation, we investigated the effect of different medium perfusion frequencies on cell reorganization and the expression of the early cardiac mesoderm reporter MESP1mCherry by live-cell imaging. Our study demonstrates that microfluidic technology can be used to automatically culture, differentiate and study hESC in very low-volume culture chambers even without continuous medium perfusion. This result is an important step towards further automation and parallelization in stem cell technology.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness.

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

Automated image analysis system for studying cardiotoxicity in human pluripotent stem cell-Derived cardiomyocytes.

BACKGROUND: Cardiotoxicity, characterized by severe cardiac dysfunction, is a major problem in patients treated with different classes of anticancer drugs. Development of predictable human-based models and assays for drug screening are crucial for preventing potential drug-induced adverse effects. Current animal in vivo models and cell lines are not always adequate to represent human biology. Alternatively, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show great potential for disease modelling and drug-induced toxicity screenings. Fully automated high-throughput screening of drug toxicity on hiPSC-CMs by fluorescence image analysis is, however, very challenging, due to clustered cell growth patterns and strong intracellular and intercellular variation in the expression of fluorescent markers. RESULTS: In this paper, we report on the development of a fully automated image analysis system for quantification of cardiotoxic phenotypes from hiPSC-CMs that are treated with various concentrations of anticancer drugs doxorubicin or crizotinib. This high-throughput system relies on single-cell segmentation by nuclear signal extraction, fuzzy C-mean clustering of cardiac α-actinin signal, and finally nuclear signal propagation. When compared to manual segmentation, it generates precision and recall scores of 0.81 and 0.93, respectively. CONCLUSIONS: Our results show that our fully automated image analysis system can reliably segment cardiomyocytes even with heterogeneous α-actinin signals.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

Transcriptome of human foetal heart compared with cardiomyocytes from pluripotent stem cells.

Differentiated derivatives of human pluripotent stem cells (hPSCs) are often considered immature because they resemble foetal cells more than adult, with hPSC-derived cardiomyocytes (hPSC-CMs) being no exception. Many functional features of these cardiomyocytes, such as their cell morphology, electrophysiological characteristics, sarcomere organization and contraction force, are underdeveloped compared with adult cardiomyocytes. However, relatively little is known about how their gene expression profiles compare with the human foetal heart, in part because of the paucity of data on the human foetal heart at different stages of development. Here, we collected samples of matched ventricles and atria from human foetuses during the first and second trimester of development. This presented a rare opportunity to perform gene expression analysis on the individual chambers of the heart at various stages of development, allowing us to identify not only genes involved in the formation of the heart, but also specific genes upregulated in each of the four chambers and at different stages of development. The data showed that hPSC-CMs had a gene expression profile similar to first trimester foetal heart, but after culture in conditions shown previously to induce maturation, they cluster closer to the second trimester foetal heart samples. In summary, we demonstrate how the gene expression profiles of human foetal heart samples can be used for benchmarking hPSC-CMs and also contribute to determining their equivalent stage of development.

Concise Review: Fluorescent Reporters in Human Pluripotent Stem Cells: Contributions to Cardiac Differentiation and Their Applications in Cardiac Disease and Toxicity.

In the last decade, since the first report of induced pluripotent stem cells, the stem cell field has made remarkable progress in the differentiation to specialized cell-types of various tissues and organs, including the heart. Cardiac lineage- and tissue-specific human pluripotent stem cell (hPSC) reporter lines have been valuable for the identification, selection, and expansion of cardiac progenitor cells and their derivatives, and for our current understanding of the underlying molecular mechanisms. In order to further advance the use of hPSCs in the fields of regenerative medicine, disease modeling, and preclinical drug development in cardiovascular research, it is crucial to identify functionally distinct cardiac subtypes and to study their biological signaling events and functional aspects in healthy and diseased conditions. In this review, we discuss the various strategies that have been followed to generate and study fluorescent reporter lines in hPSCs and provide insights how these reporter lines contribute to a better understanding and improvement of cell-based therapies and preclinical drug and toxicity screenings in the cardiac field.

Generation and purification of human stem cell-derived cardiomyocytes.

Efficient and reproducible generation and purification of human stem cell-derived cardiomyocytes (CMs) is crucial for regenerative medicine, disease modeling, drug screening and study of developmental events during cardiac specification. Established methods to generate CMs from human pluripotent stem cells (hPSCs) include the Spin-embryoid body (Spin-EB) and monolayer-based differentiation protocol. In the presence of an optimized cocktail of growth factors under defined conditions, hPSCs differentiate efficiently into functional contracting CMs within 10 days. Nevertheless, despite high efficiencies, cardiac-directed differentiations of hPSCs typically result in heterogeneous populations comprised of both CMs and uncharacterized non-cardiac cell-types. Therefore, generation of pure populations of stem cell-derived CMs is of fundamental importance for basic cardiac research and pre-clinical and possible clinical applications. For the purification of CMs from heterogeneous populations, fluorescent activated cell sorting (FACS) is a widely appreciated method. Nonetheless, FACS-based isolation of CMs comes along with several disadvantages, such as undesired contaminations and low viability of target cells. Here, we describe a convenient and rapid procedure for the purification of hPSCs-derived CMs under sterile culture conditions, resulting in high purity and viability of sorted CMs. Purification with VCAMI-coupled magnetic Dynabeads led to robust enrichment of CMs, which will especially be important for cardiac differentiations of cell lines with poor differentiation efficiencies. In addition, this will also be beneficial for the standardization and reproducibility of human stem cell-derived assays in the fields of cardiac disease modeling, drug discovery and disease modeling.

Dual reporter MESP1 mCherry/w-NKX2-5 eGFP/w hESCs enable studying early human cardiac differentiation.

Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1(mCherry/w)-NKX2-5(eGFP/w) reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-α, CD13, and ROR-2. No enrichment was found for the previously described KDR+PDGFR-α+ progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.

NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

Stem-cell-based therapy and lessons from the heart.

The potential usefulness of human embryonic stem cells for therapy derives from their ability to form any cell in the body. This potential has been used to justify intensive research despite some ethical concerns. In parallel, scientists have searched for adult stem cells that can be used as an alternative to embryonic cells, and, for the heart at least, these efforts have led to promising results. However, most adult cardiomyocytes are unable to divide and form new cardiomyocytes and would therefore be unable to replace those lost as a result of disease. Basic questions--for example, whether cardiomyocyte replacement or alternatives, such as providing the damaged heart with new blood vessels or growth factors to activate resident stem cells, are the best approach--remain to be fully addressed. Despite this, preclinical studies on cardiomyocyte transplantation in animals and the first clinical trials with adult stem cells have recently been published with mixed results.

Micro-Engineered Heart Tissues On-Chip with Heterotypic Cell Composition Display Self-Organization and Improved Cardiac Function.

Advanced in vitro models that recapitulate the structural organization and function of the human heart are highly needed for accurate disease modeling, more predictable drug screening, and safety pharmacology. Conventional 3D Engineered Heart Tissues (EHTs) lack heterotypic cell complexity and culture under flow, whereas microfluidic Heart-on-Chip (HoC) models in general lack the 3D configuration and accurate contractile readouts. In this study, an innovative and user-friendly HoC model is developed to overcome these limitations, by culturing human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs), endothelial (ECs)- and smooth muscle cells (SMCs), together with human cardiac fibroblasts (FBs), underflow, leading to self-organized miniaturized micro-EHTs (µEHTs) with a CM-EC interface reminiscent of the physiological capillary lining. µEHTs cultured under flow display enhanced contractile performance and conduction velocity. In addition, the presence of the EC layer altered drug responses in µEHT contraction. This observation suggests a potential barrier-like function of ECs, which may affect the availability of drugs to the CMs. These cardiac models with increased physiological complexity, will pave the way to screen for therapeutic targets and predict drug efficacy.

A safety screening platform for individualized cardiotoxicity assessment.

Cardiotoxicity remains a major cause of drug withdrawal, partially due to lacking predictability of animal models. Additionally, risk of cardiotoxicity following treatment of cancer patients is treatment limiting. It is unclear which patients will develop heart failure following therapy. Human pluripotent stem cell (hPSC)-derived cardiomyocytes present an unlimited cell source and may offer individualized solutions to this problem. We developed a platform to predict molecular and functional aspects of cardiotoxicity. Our platform can discriminate between the different cardiotoxic mechanisms of existing and novel anthracyclines Doxorubicin, Aclarubicin, and Amrubicin. Doxorubicin and Aclarubicin unlike Amrubicin substantially affected the transcriptome, mitochondrial membrane integrity, contractile force and transcription factor availability. Cardiomyocytes recovered fully within two or three weeks, corresponding to the intermittent clinical treatment regimen. Our system permits the study of hPSC-cardiomyocyte recovery and the effects of accumulated dose after multiple dosing, allowing individualized cardiotoxicity evaluation, which effects millions of cancer patients treated annually.

Towards Improved Human In Vitro Models for Cardiac Arrhythmia: Disease Mechanisms, Treatment, and Models of Atrial Fibrillation.

Heart rhythm disorders, arrhythmias, place a huge economic burden on society and have a large impact on the quality of life of a vast number of people. Arrhythmias can have genetic causes but primarily arise from heart tissue remodeling during aging or heart disease. As current therapies do not address the causes of arrhythmias but only manage the symptoms, it is of paramount importance to generate innovative test models and platforms for gaining knowledge about the underlying disease mechanisms which are compatible with drug screening. In this review, we outline the most important features of atrial fibrillation (AFib), the most common cardiac arrhythmia. We will discuss the epidemiology, risk factors, underlying causes, and present therapies of AFib, as well as the shortcomings and opportunities of current models for cardiac arrhythmia, including animal models, in silico and in vitro models utilizing human pluripotent stem cell (hPSC)-derived cardiomyocytes.

Automated assessment of human engineered heart tissues using deep learning and template matching for segmentation and tracking.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

Methacrylated human recombinant collagen peptide as a hydrogel for manipulating and monitoring stiffness-related cardiac cell behavior.

Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules.

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.

CHAP is a newly identified Z-disc protein essential for heart and skeletal muscle function.

In recent years, the perception of Z-disc function has changed from a passive anchor for myofilaments that allows transmission of force, to a dynamic multicomplex structure, capable of sensing and transducing extracellular signals. Here, we describe a new Z-disc protein, which we named CHAP (cytoskeletal heart-enriched actin-associated protein), expressed in differentiating heart and skeletal muscle in vitro and in vivo. Interestingly, in addition to its sarcomeric localization, CHAP was also able to translocate to the nucleus. CHAP was associated with filamentous actin in the cytoplasm and the nucleus when expressed ectopically in vitro, but in rat neonatal cardiomyocytes, CHAP disrupted the subcellular localization of alpha-actinin, another Z-disc protein. More importantly, knockdown of CHAP in zebrafish resulted in aberrant cardiac and skeletal muscle development and function. These findings suggest that CHAP is a critical component of the sarcomere with an important role in muscle development.