A Scalable Approach Reveals Functional Responses of iPSC Cardiomyocyte 3D Spheroids

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) provide an in vitro model of the human myocardium. Complex 3D scaffolded culture methods improve the phenotypical maturity of iPSC-CMs, although typically at the expense of throughput. We have developed a novel, scalable approach that enables the use of iPSC-CM 3D spheroid models in a label-free readout system in a standard 96-well plate-based format. Spheroids were accurately positioned onto recording electrodes using a magnetic gold–iron oxide nanoparticle approach. Remarkably, both contractility (impedance) and extracellular field potentials (EFPs) could be detected from the actively beating spheroids over long durations and after automated dosing with pharmacological agents. The effects on these parameters of factors, such as co-culture (including human primary cardiac fibroblasts), extracellular buffer composition, and electrical pacing, were investigated. Beat amplitudes were increased greater than 15-fold by co-culture with fibroblasts. Optimization of extracellular Ca2+ fluxes and electrical pacing promoted the proper physiological response to positive inotropic agonists of increased beat amplitude (force) rather than the increased beat rate often observed in iPSC-CM studies. Mechanistically divergent repolarizations in different spheroid models were indicated by their responses to BaCl2 compared with E-4031. These studies demonstrate a new method that enables the pharmacological responses of 3D iPSC-CM spheroids to be determined in a label-free, standardized, 96-well plate-based system. This approach could have discovery applications across cardiovascular efficacy and safety, where parameters typically sought as readouts of iPSC-CM maturity or physiological relevance have the potential to improve assay predictivity.

Download Full-text

Human Cardiac Organoids for Modeling Genetic Cardiomyopathy

Cells ◽

10.3390/cells9071733 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1733 ◽

Cited By ~ 1

Author(s):

Michele Filippo Buono ◽

Lisa von Boehmer ◽

Jaan Strang ◽

Simon P. Hoerstrup ◽

Maximilian Y. Emmert ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Promising Tool ◽

Vitro Model ◽

Human Cardiac Fibroblasts ◽

Induced Pluripotent ◽

First Time ◽

Design And Testing

Genetic cardiomyopathies are characterized by changes in the function and structure of the myocardium. The development of a novel in vitro model could help to better emulate healthy and diseased human heart conditions and may improve the understanding of disease mechanisms. In this study, for the first time, we demonstrated the generation of cardiac organoids using a triculture approach of human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs)—from healthy subjects and cardiomyopathy patients—human cardiac microvascular endothelial cells (HCMECs) and human cardiac fibroblasts (HCFs). We assessed the organoids’ suitability as a 3D cellular model for the representation of phenotypical features of healthy and cardiomyopathic hearts. We observed clear differences in structure and beating behavior between the organoid groups, depending on the type of hiPS-CMs (healthy versus cardiomyopathic) used. Organoids may thus prove a promising tool for the design and testing of patient-specific treatments as well as provide a platform for safer and more efficacious drug development.

Download Full-text

Engineering aligned human cardiac muscle using developmentally inspired fibronectin micropatterns

Scientific Reports ◽

10.1038/s41598-021-87550-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ivan Batalov ◽

Quentin Jallerat ◽

Sean Kim ◽

Jacqueline Bliley ◽

Adam W. Feinberg

Keyword(s):

Cardiac Fibroblasts ◽

Induced Pluripotent Stem Cell ◽

Confocal Imaging ◽

Embryonic Chick ◽

Cardiac Tissues ◽

Density Dependent ◽

Induced Pluripotent ◽

Cell Cell

AbstractCardiac two-dimensional tissues were engineered using biomimetic micropatterns based on the fibronectin-rich extracellular matrix (ECM) of the embryonic heart. The goal of this developmentally-inspired, in vitro approach was to identify cell–cell and cell-ECM interactions in the microenvironment of the early 4-chambered vertebrate heart that drive cardiomyocyte organization and alignment. To test this, biomimetic micropatterns based on confocal imaging of fibronectin in embryonic chick myocardium were created and compared to control micropatterns designed with 2 or 20 µm wide fibronectin lines. Results show that embryonic chick cardiomyocytes have a unique density-dependent alignment on the biomimetic micropattern that is mediated in part by N-cadherin, suggesting that both cell–cell and cell-ECM interactions play an important role in the formation of aligned myocardium. Human induced pluripotent stem cell-derived cardiomyocytes also showed density-dependent alignment on the biomimetic micropattern but were overall less well organized. Interestingly, the addition of human adult cardiac fibroblasts and conditioning with T3 hormone were both shown to increase human cardiomyocyte alignment. In total, these results show that cardiomyocyte maturation state, cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblast interactions, and cardiomyocyte-ECM interactions can all play a role when engineering anisotropic cardiac tissues in vitro and provides insight as to how these factors may influence cardiogenesis in vivo.

Download Full-text

Generation of Quiescent Cardiac Fibroblasts From Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis

Circulation Research ◽

10.1161/circresaha.119.315491 ◽

2019 ◽

Vol 125 (5) ◽

pp. 552-566 ◽

Cited By ~ 20

Author(s):

Hao Zhang ◽

Lei Tian ◽

Mengcheng Shen ◽

Chengyi Tu ◽

Haodi Wu ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

In Vitro Modeling ◽

Induced Pluripotent

Download Full-text

The Combination of Cell Cultured Technology and In Silico Model to Inform the Drug Development

Pharmaceutics ◽

10.3390/pharmaceutics13050704 ◽

2021 ◽

Vol 13 (5) ◽

pp. 704

Author(s):

Zhengying Zhou ◽

Jinwei Zhu ◽

Muhan Jiang ◽

Lan Sang ◽

Kun Hao ◽

...

Keyword(s):

Drug Development ◽

In Silico ◽

In Vitro Models ◽

Clinical Settings ◽

Culture Methods ◽

Physiologically Based Pharmacokinetic ◽

Induced Pluripotent ◽

In Silico Models

Human-derived in vitro models can provide high-throughput efficacy and toxicity data without a species gap in drug development. Challenges are still encountered regarding the full utilisation of massive data in clinical settings. The lack of translated methods hinders the reliable prediction of clinical outcomes. Therefore, in this study, in silico models were proposed to tackle these obstacles from in vitro to in vivo translation, and the current major cell culture methods were introduced, such as human-induced pluripotent stem cells (hiPSCs), 3D cells, organoids, and microphysiological systems (MPS). Furthermore, the role and applications of several in silico models were summarised, including the physiologically based pharmacokinetic model (PBPK), pharmacokinetic/pharmacodynamic model (PK/PD), quantitative systems pharmacology model (QSP), and virtual clinical trials. These credible translation cases will provide templates for subsequent in vitro to in vivo translation. We believe that synergising high-quality in vitro data with existing models can better guide drug development and clinical use.

Download Full-text

Tropomyosin 1 Genetically Constrains in Vitro Megakaryopoiesis

Blood ◽

10.1182/blood-2019-121538 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3612-3612

Author(s):

Christopher Thom ◽

Chintan Jobaliya ◽

Kimberly Lorenz ◽

Jean Ann Maguire ◽

Alyssa Gagne ◽

...

Keyword(s):

Gene Expression ◽

Machine Learning ◽

Expression Patterns ◽

Genome Wide Association ◽

Quantitative Prediction ◽

Genome Wide Association Studies ◽

Functional Responses ◽

Genome Wide ◽

Induced Pluripotent

Platelet size and count are heritable, quantifiable outcomes of hematopoiesis. A better understanding of genetic mechanisms that regulate hematopoiesis could yield insights into blood disorders and augment clinical translational efforts to generate platelets in vitro. For example, induced pluripotent stem cell-derived blood cells could someday ameliorate adverse effects and increase transfusable platelet supplies. However, these methods remain inefficient. Genome wide association studies (GWAS) have linked hundreds of DNA loci with altered human platelet traits, but little is known about how these GWAS loci mechanistically impact hematopoiesis, megakaryocyte or platelet biology. Using publicly available genome-wide association and epigenetic data sets, we applied a machine-learning framework to identify epigenetic features enriched at established platelet trait association sites. From these results, we derived a quantitative prediction model that identified hematopoiesis-, megakaryocyte-, and platelet-relevant genomic loci and related genes more accurately than any prior computational method. In addition to specifying exact genetic variants known to regulate platelet traits and function, our model highlighted several novel variants in established platelet trait variation GWAS loci that were predicted to disrupt key transcription factor binding sites. Among loci nominated by our statistical model was a variant (rs11071720) that alters Tropomyosin 1 (TPM1) gene expression. TPM1 regulates cytoskeletal biology in many cell types, and cytoskeletal functions critically impact hematopoiesis and terminal blood cell biology. We used CRISPR/Cas9-mediated genome editing to create TPM1-knockout human induced pluripotent stem cells. TPM1KO cells were healthy and showed normal developmental progression through primitive streak, mesoderm, and early primitive hematopoietic differentiation programs. Unexpectedly, TPM1KO cells demonstrated enhanced formation of hemogenic endothelium and hematopoietic progenitor cells, increasing total megakaryocyte yield by more than 2-fold. TPM1KO megakaryocytes demonstrated normal morphology, gene expression patterns and functional responses to platelet agonists. Our findings help explain human genetics associations and identify a novel strategy to enhance in vitro hematopoiesis. More broadly, our now-validated machine learning-based approach might specify additional functional loci underlying hematopoiesis, megakaryopoiesis, or thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery

Toxicological Sciences ◽

10.1093/toxsci/kfz168 ◽

2019 ◽

Vol 172 (1) ◽

pp. 89-97 ◽

Cited By ~ 10

Author(s):

Nicole T Feric ◽

Isabella Pallotta ◽

Rishabh Singh ◽

Danielle R Bogdanowicz ◽

Marietta M Gustilo ◽

...

Keyword(s):

Drug Discovery ◽

Cardiac Fibroblasts ◽

Cardiac Contractility ◽

Cardiac Cells ◽

Force Frequency ◽

Cardiac Tissues ◽

Omecamtiv Mecarbil ◽

Induced Pluripotent ◽

Force Frequency Response

Abstract Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the β-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

Download Full-text

Haemostatic Platelet Plug Formation in the Isolated Rabbit Mesenteric Preparation - An Analysis of Red Blood Cell Participation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650069 ◽

1980 ◽

Vol 44 (01) ◽

pp. 006-008 ◽

Cited By ~ 12

Author(s):

D Bergqvist ◽

K-E Arfors

Keyword(s):

Whole Blood ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

In Vitro Test ◽

Pharmacological Agents ◽

Vitro Test ◽

Releasing Effect ◽

Plug Formation

SummaryIn a model using an isolated rabbit mesenteric preparation microvessels were transected and the time until haemostatic plugs formed was registered. Perfusion of platelet rich plasma gave no haemostasis whereas whole blood did. Addition of chlorpromazine or adenosine to the whole blood significantly prolonged the time for haemostasis, and addition of ADP to the platelet rich plasma significantly shortened it. It is concluded that red cells are necessary for a normal haemostasis in this model, probably by a combination of a haemodynamic and ADP releasing effect.The fundamental role of platelets in haemostatic plug formation is unquestionable but there are still problems concerning the stimulus for this process to start. Three platelet aggregating substances have been discussed – thrombin, adenosine diphosphate (ADP) and collagen. Evidence speaking in favour of thrombin is, however, very minimal, and the discussion has to be focused on collagen and ADP. In an in vitro system using polyethylene tubings we have shown that "haemostasis" can be obtained without the presence of collagen but against these results can be argued that it is only another in vitro test for platelet aggregation (1).To be able to induce haemostasis in this model, however, the presence of red blood cells is necessary. To further study this problem we have developed a model where haemostatic plug formation can be studied in the isolated rabbit mesentery and we have briefly reported on this (2).Thus, it is possible to perfuse the vessels with whole blood as well as with platelet rich plasma (PRP) and different pharmacological agents of importance.

Download Full-text