Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both ...in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.
Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the ...molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.
Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study ...of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.
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•Reprogramming and cardiac differentiation preserve patient-specific gene expression•Metabolic and stress genes account for inter-patient transcriptome variation•Bioinformatics analysis predicts patient-specific drug-induced cardiotoxicity•Drug-induced cardiotoxicity can be functionally evaluated in vitro using hiPSC-CMs
hiPSC-CM transcriptome profiling showed greater inter-patient than intra-patient variation. Toxicology analysis predicted and functionally validated individualized drug responsiveness, suggesting that hiPSC-CMs could serve as preclinical readout platforms for precision medicine.
Abstract Background Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic ...control. Objectives This study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control. Methods The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology. Results Patch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future. Conclusions Our study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.
Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and ...progression to heart failure.
Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.
We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.
EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we ...established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
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•β-AR signaling switch from β-2 AR to β-1/2 AR mode during human iPSC-CM maturation•Upregulation of PDE2/3 leads to compromised β-adrenergic regulation in DCM iPSC-CMs•Epigenetic activation of PDE2/3 is a key molecular event during pathogenesis of DCM•Nuclear localization of mutated TNNT2 contributes to epigenetic modification in DCM
In this paper, Wu et al. profiled the β-adrenergic signaling properties in human iPSC-CMs and demonstrated novel epigenetic mechanisms that underlie the compromised β-adrenergic signaling in DCM, a common cause of heart failure and cardiac transplantation. These results enhance our understanding of DCM pathogenesis and may uncover new therapeutic targets.
The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. ...Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin
flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 10
hPSC-CMs were mixed with 0.4 × 10
human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.
Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in ...cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes.
To demonstrate intact neuregulin (NRG)-1β/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures.
All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1β, an anti-NRG-1β neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1β/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype.
NRG-1β/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1β/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker.
Cell therapies offer the potential to improve cardiac function after myocardial infarction. Although injection of single-cell suspensions has proven safe, cell retention and survival rates are low. ...Tissue-engineered grafts allow cell delivery with minimal initial cell loss and mechanical support to the heart. However, graft performance cannot be easily compared, and optimal construct thickness, vascularization, and survival kinetics are unknown.
Cardiac tissue slices (CTS) were generated by sectioning mouse hearts (n=40) expressing firefly luciferase and green fluorescent protein into slices of defined size and thickness using a vibrating blade microtome. Bioluminescence imaging of CTS transplanted onto hearts of immunodeficient mice demonstrated survival of ≤30% of transplanted cells. Cardiac slice perfusion was re-established within 3 days, likely through anastomosis of pre-existing vessels with the host vasculature and invasion of vessels from the host. Immunofluorescence showed a peak in cell death 3 days after transplantation and a gradual decline thereafter. MRI revealed preservation of contractile function and an improved ejection fraction 1 month after transplantation of CTS (28±2% CTS versus 22±2% control; P=0.05). Importantly, this effect was specific to CTS because transplantation of skeletal muscle tissue slices led to faster dilative remodeling and higher animal mortality.
In summary, this is the first study to use CTS as a benchmark to validate and model tissue-engineered graft studies. CTS transplantation improved cell survival, established reperfusion, and enhanced cardiac function after myocardial infarction. These findings also confirm that dilative remodeling can be attenuated by topical transplantation of CTS but not skeletal muscle tissue grafts.
Previous studies have shown that prolonged propagation of undifferentiated human embryonic stem cells (hESCs) requires conditioned medium from mouse embryonic feeders (MEF-CM) as well as matrix ...components. Because hESCs express growth factor receptors, including those for basic fibroblast growth factor (bFGF), stem cell factor (SCF), and fetal liver tyrosine kinase-3 ligand (Flt3L), we evaluated these and other growth factors for their ability to maintain undifferentiated hESCs in the absence of conditioned medium. We found cultures maintained in bFGF alone or in combination with other factors showed characteristics similar to MEF-CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In contrast, cells in media containing Flt-3L, thrombopoietin, and SCF, individually or in combination, showed almost complete differentiation after 6 weeks in culture. These data demonstrate that hESCs can be maintained in nonconditioned medium using growth factors.
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