Abnormal developmental trajectory and vulnerability to cardiac arrhythmias in tetralogy of Fallot with DiGeorge syndrome.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Ventricular dysfunction and cardiac arrhythmias are well-documented complications in patients with repaired TOF. Whether intrinsic abnormalities exist in TOF cardiomyocytes is unknown. We establish human induced pluripotent stem cells (hiPSCs) from TOF patients with and without DiGeorge (DG) syndrome, the latter being the most commonly associated syndromal association of TOF. TOF-DG hiPSC-derived cardiomyocytes (hiPSC-CMs) show impaired ventricular specification, downregulated cardiac gene expression and upregulated neural gene expression. Transcriptomic profiling of the in vitro cardiac progenitors reveals early bifurcation, as marked by ectopic RGS13 expression, in the trajectory of TOF-DG-hiPSC cardiac differentiation. Functional assessments further reveal increased arrhythmogenicity in TOF-DG-hiPSC-CMs. These findings are found only in the TOF-DG but not TOF-with no DG (ND) patient-derived hiPSC-CMs and cardiac progenitors (CPs), which have implications on the worse clinical outcomes of TOF-DG patients.

APLNR marks a cardiac progenitor derived with human induced pluripotent stem cells.

Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration.

Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues.

Cardiac in vitro models have become increasingly obtainable and affordable with the optimization of human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) differentiation. However, these CMs are immature compared to their in vivo counterparts. Here we study the cellular phenotype of hPSC-CMs by comparing their single-cell gene expression and functional profiles in three engineered cardiac tissue configurations: human ventricular (hv) cardiac anisotropic sheet, cardiac tissue strip, and cardiac organoid chamber (hvCOC), with spontaneously aggregated 3D cardiac spheroids (CS) as control. The CM maturity was found to increase with increasing levels of complexity of the engineered tissues from CS to hvCOC. The contractile components are the first function to mature, followed by electrophysiology and oxidative metabolism. Notably, the 2D tissue constructs show a higher cellular organization whereas metabolic maturity preferentially increases in the 3D constructs. We conclude that the tissue engineering models resembling configurations of native tissues may be reliable for drug screening or disease modeling.

Arrhythmic Risk Assessment of Hypokalaemia Using Human Pluripotent Stem Cell-Derived Cardiac Anisotropic Sheets.

Introduction: Hypokalaemia, defined as an extracellular concentration of K+ below 3.5 mM, can cause cardiac arrhythmias by triggered or re-entrant mechanisms. Whilst these effects have been reported in animal and human stem cell-based models, to date there has been no investigation in more complex structures such as the human ventricular cardiac anisotropic sheet (hvCAS). Here, we investigated arrhythmogenicity, electrophysiological, and calcium transient (CaT) changes induced by hypokalaemia using this bioengineered platform. Methods: An optical mapping technique was applied on hvCAS derived from human pluripotent stem cells to visualize electrophysiological and CaT changes under normokalaemic (5 mM KCl) and hypokalaemic (3 mM KCl) conditions. Results: Hypokalaemia significantly increased the proportion of preparations showing spontaneous arrhythmias from 0/14 to 7/14 (Fisher's exact test, p = 0.003). Hypokalaemia reduced longitudinal conduction velocity (CV) from 7.81 to 7.18 cm⋅s-1 (n = 9, 7; p = 0.036), transverse CV from 5.72 to 4.69 cm⋅s-1 (n = 12, 11; p = 0.030), prolonged action potential at 90% repolarization (APD90) from 83.46 to 97.45 ms (n = 13, 15; p < 0.001), increased action potential amplitude from 0.888 to 1.195 ΔF (n = 12, 14; p < 0.001) and CaT amplitude from 0.76 to 1.37 ΔF (n = 12, 13; p < 0.001), and shortened effective refractory periods from 242 to 165 ms (n = 12, 13; p < 0.001). Conclusion: Hypokalaemia exerts pro-arrhythmic effects on hvCAS, which are associated with alterations in CV, repolarization, refractoriness, and calcium handling. These preparations provide a useful platform for investigating electrophysiological substrates and for conducting arrhythmia screening.

Human Pluripotent Stem Cells for Modeling of Anticancer Therapy-Induced Cardiotoxicity and Cardioprotective Drug Discovery.

Anticancer chemotherapies have been shown to produce severe side effects, with cardiotoxicity from anthracycline being the most notable. Identifying risk factors for anticancer therapy-induced cardiotoxicity in cancer patients as well as understanding its underlying mechanism is essential to improving clinical outcomes of chemotherapy treatment regimens. Moreover, cardioprotective agents against anticancer therapy-induced cardiotoxicity are scarce. Human induced pluripotent stem cell technology offers an attractive platform for validation of potential single nucleotide polymorphism with increased risk for cardiotoxicity. Successful validation of risk factors and mechanism of cardiotoxicity would aid the development of such platform for novel drug discovery and facilitate the practice of personalized medicine.

Circulating high-sensitivity troponin T and microRNAs as markers of myocardial damage during childhood leukaemia treatment.

BACKGROUND: We investigated whether plasma high-sensitivity cardiac troponin T (hs-cTnT) and circulating heart-associated microRNA (miRs) are increased in children with leukaemias during anthracycline-based chemotherapeutic treatment. METHODS: In vitro human pluripotent stem cell (hPSC)-derived cardiomyocyte model showed that miR-1, miR-133a, miR-208a, miR-208b, and miR-499 are released from cells into culture medium in a time- and dose-dependent manner on doxorubicin exposure. Left ventricular (LV) myocardial deformation and circulating heart-associated miRs and plasma hs-cTnT during and after completion of chemotherapy were determined in 40 children with newly diagnosed acute leukaemia. RESULTS: Significant reduction of LV global longitudinal strain and strain rates were found within 1 week after completion of anthracycline therapy in the induction phase of treatment (all p < 0.05). Hs-cTnT level peaked and miR-1 increased significantly at this time point. Log-transformed hs-cTnT correlated negatively with LV global systolic longitudinal strain (r = -0.38, p < 0.001). Receiver operating characteristic analysis revealed that area under the curve for changes in plasma hs-cTnT from baseline and plasma miR-1 levels in detecting a reduction in ≥20% of global longitudinal strain were respectively 0.62 (95% CI 0.38-0.87) and 0.62 (95% CI 0.40-0.84). CONCLUSION: Plasma hs-cTnT and circulating miR-1 may be useful markers of myocardial damage during chemotherapy in children with leukaemias. IMPACT: Heart-associated miRNAs including miR-1, miR-133a, miR-208a, miR-208b,and miR-499 were increased in the culture medium upon exposure of hPSC-derived cardiomyocytes to doxorubicin. Only miR-1 increased significantly during anthracycline-based therapy in paediatric leukaemic patients. In paediatric leukaemic patients, plasma hs-cTnT and circulating level of miR-1 showed the most significant increase within 1 week after completion of anthracycline therapy in the induction treatment phase. The study provides the first evidence of progressive increase in circulating miR-1 and plasma hs-cTnT levels during the course of anthracycline-based therapy in children with leukaemias, with hs-cTnT level also associated with changes in LV myocardial deformation.

A Heart-Breast Cancer-on-a-Chip Platform for Disease Modeling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-ß 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

Single-Cell Transcriptomics of Engineered Cardiac Tissues From Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Abnormal Developmental Trajectory and Intrinsic Contractile Defects in Hypoplastic Right Heart Syndrome.

Background To understand the intrinsic cardiac developmental and functional abnormalities in pulmonary atresia with intact ventricular septum (PAIVS) free from effects secondary to anatomic defects, we performed and compared single-cell transcriptomic and phenotypic analyses of patient- and healthy subject-derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and engineered tissue models. Methods and Results We derived hiPSC lines from 3 patients with PAIVS and 3 healthy subjects and differentiated them into hiPSC-CMs, which were then bioengineered into the human cardiac anisotropic sheet and human cardiac tissue strip custom-designed for electrophysiological and contractile assessments, respectively. Single-cell RNA sequencing (scRNA-seq) of hiPSC-CMs, human cardiac anisotropic sheet, and human cardiac tissue strip was performed to examine the transcriptomic basis for any phenotypic abnormalities using pseudotime and differential expression analyses. Through pseudotime analysis, we demonstrated that bioengineered tissue constructs provide pro-maturational cues to hiPSC-CMs, although the maturation and development were attenuated in PAIVS hiPSC-CMs. Furthermore, reduced contractility and prolonged contractile kinetics were observed with PAIVS human cardiac tissue strips. Consistently, single-cell RNA sequencing of PAIVS human cardiac tissue strips and hiPSC-CMs exhibited diminished expression of cardiac contractile apparatus genes. By contrast, electrophysiological aberrancies were absent in PAIVS human cardiac anisotropic sheets. Conclusions Our findings were the first to reveal intrinsic abnormalities of cardiomyocyte development and function in PAIVS free from secondary effects. We conclude that hiPSC-derived engineered tissues offer a unique method for studying primary cardiac abnormalities and uncovering pathogenic mechanisms that underlie sporadic congenital heart diseases.

Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium-on-a-Chip Application.

Bioprinting holds great promise towards engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, we have developed a novel and mechanically robust biomaterial-ink based on non-mulberry silk fibroin protein. The silk-based ink demonstrated suitable mechanical properties required in terms of elasticity and stiffness (~40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allowed the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which were able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin-43, and maintain synchronously beating of cardiomyocytes. The printed constructs were found to be non-immunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue-on-a-chip, the silk-based ink was used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC-CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. We confirmed the ability of silk-based supporting hydrogel towards maturation and viability of hiPSC-CSs and endothelial cells, and for applications in evaluating drug toxicity.

Combinatorial Treatment of Human Cardiac Engineered Tissues With Biomimetic Cues Induces Functional Maturation as Revealed by Optical Mapping of Action Potentials and Calcium Transients.

Although biomimetic stimuli, such as microgroove-induced alignment (µ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined µ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-ß signaling was downregulated; the TGF-ß receptor agonist and antagonist TGF-ß1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.

Sarco/endoplasmic reticulum Ca2+-ATPase is a more effective calcium remover than sodium-calcium exchanger in human embryonic stem cell-derived cardiomyocytes.

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+-handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights.NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.

Structural and Mechanistic Bases of Nuclear Calcium Signaling in Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes.

The loss of nonregenerative, terminally differentiated cardiomyocytes (CMs) due to aging or diseases is generally considered irreversible. Human pluripotent stem cells (hPSCs) can self-renew while maintaining their pluripotency to differentiate into all cell types, including ventricular (V) cardiomyocytes (CMs), to provide a potential unlimited ex vivo source of CMs for heart disease modeling, drug/cardiotoxicity screening, and cell-based therapies. In the human heart, cytosolic Ca2+ signals are well characterized but the contribution of nuclear Ca2+ is essentially unexplored. The present study investigated nuclear Ca2+ signaling in hPSC-VCMs. Calcium transient or sparks in hPSC-VCMs were measured by line scanning using a spinning disc confocal microscope. We observed that nuclear Ca2+, which stems from unitary sparks due to the diffusion of cytosolic Ca2+ that are mediated by RyRs on the nuclear reticulum, is functional. Parvalbumin- (PV-) mediated Ca2+ buffering successfully manipulated Ca2+ transient and stimuli-induced apoptosis in hPSC-VCMs. We also investigated the effect of Ca2+ on gene transcription in hPSC-VCMs, and the involvement of nuclear factor of activated T-cell (NFAT) pathway was identified. The overexpression of Ca2+-sensitive, nuclear localized Ca2+/calmodulin-dependent protein kinase II δ B (CaMKIIδ B) induced cardiac hypertrophy through nuclear Ca2+/CaMKIIδB/HDAC4/MEF2 pathway. These findings provide insights into nuclear Ca2+ signal in hPSC-VCMs, which may lead to novel strategies for maturation as well as improved systems for disease modeling, drug discovery, and cell-based therapies.

Modulation of chromatin remodeling proteins SMYD1 and SMARCD1 promotes contractile function of human pluripotent stem cell-derived ventricular cardiomyocyte in 3D-engineered cardiac tissues.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of differentiating into functional cardiomyocytes (CMs) for cell replacement therapy, tissue engineering, drug discovery and toxicity screening. From a scale-free, co-expression network analysis of transcriptomic data that distinguished gene expression profiles of undifferentiated hESC, hESC-, fetal- and adult-ventricular(V) CM, two candidate chromatin remodeling proteins, SMYD1 and SMARCD1 were found to be differentially expressed. Using lentiviral transduction, SMYD1 and SMARCD1 were over-expressed and suppressed, respectively, in single hESC-VCMs as well as the 3D constructs Cardiac Micro Tissues (CMT) and Tissue Strips (CTS) to mirror the endogenous patterns, followed by dissection of their roles in controlling cardiac gene expression, contractility, Ca2+-handling, electrophysiological functions and in vitro maturation. Interestingly, compared to independent single transductions, simultaneous SMYD1 overexpression and SMARCD1 suppression in hESC-VCMs synergistically interacted to increase the contractile forces of CMTs and CTSs with up-regulated transcripts for cardiac contractile, Ca2+-handing, and ion channel proteins. Certain effects that were not detected at the single-cell level could be unleashed under 3D environments. The two chromatin remodelers SMYD1 and SMARCD1 play distinct roles in cardiac development and maturation, consistent with the notion that epigenetic priming requires triggering signals such as 3D environmental cues for pro-maturation effects.

Human Cardiac Ventricular-Like Organoid Chambers and Tissue Strips From Pluripotent Stem Cells as a Two-Tiered Assay for Inotropic Responses.

Traditional drug discovery is an inefficient process. Human pluripotent stem cell-derived cardiomyocytes can potentially fill the gap between animal and clinical studies, but conventional two-dimensional cultures inadequately recapitulate the human cardiac phenotype. Here, we systematically examined the pharmacological responses of engineered human ventricular-like cardiac tissue strips (hvCTS) and organoid chambers (hvCOC) to 25 cardioactive compounds covering various drug classes. While hvCTS effectively detected negative and null inotropic effects, the sensitivity to positive inotropes was modest. We further quantified the predictive capacity of hvCTS in a blinded screening, with accuracies for negative, positive, and null inotropic effects at 100%, 86%, and 80%, respectively. Interestingly, hvCOC, with a pro-maturation milieu that yields physiologically complex parameters, displayed enhanced positive inotropy. Based on these results, we propose a two-tiered screening system for avoiding false positives and negatives. Such an approach would facilitate drug discovery by leading to better overall success.

TRPV6 protects ER stress-induced apoptosis via ATF6α-TRPV6-JNK pathway in human embryonic stem cell-derived cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes have potential applications in disease modeling and drug screening. Therefore, it is important to understand the mechanisms and signaling pathways underlying the survival and death of these cells. Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding in the ER. Cells cope with ER stress by activating the unfolded protein response (UPR), a homeostatic signaling network that orchestrates the recovery of ER function. In the present study, we hypothesized that ER stress may upregulate the expression of transient receptor potential channel TRPV6, which in turn serves to protect human embryonic stem cell-derived cardiomyocytes (hESC-CMs) from ER stress-induced apoptotic cell death. Indeed, we found that ER stress induced by thapsigargin and tunicamycin led to increased expression of TRPV6 via ATF6α signaling branch. siRNA-mediated knockdown of TRPV6 aggravated ER stress-induced apoptotic cell death, whereas overexpression of TRPV6 attenuated ER stress-induced apoptosis in hESC-CMs. Furthermore, the signaling pathway downstream of TRPV6 was MAPK-JNK. Taken together, these results provide strong evidence that, under ER stress, TRPV6 is upregulated to protect hESC-CMs from apoptotic cell death via ATF6α-TRPV6-JNK pathway.

Probing flecainide block of INa using human pluripotent stem cell-derived ventricular cardiomyocytes adapted to automated patch-clamping and 2D monolayers.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 ±â€¯11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of INa, ICaL, Ikr and If and similarly lacked Ito and IK1. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced INa blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.

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.

Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells.

Tissue engineers and stem cell biologists have made exciting progress toward creating simplified models of human heart muscles or aligned monolayers to help bridge a longstanding gap between experimental animals and clinical trials. However, no existing human in vitro systems provide the direct measures of cardiac performance as a pump. Here, we developed a next-generation in vitro biomimetic model of pumping human heart chamber, and demonstrated its capability for pharmaceutical testing. From human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCM) embedded in collagen-based extracellular matrix hydrogel, we engineered a three-dimensional (3D) electro-mechanically coupled, fluid-ejecting miniature human ventricle-like cardiac organoid chamber (hvCOC). Structural characterization showed organized sarcomeres with myofibrillar microstructures. Transcript and RNA-seq analyses revealed upregulation of key Ca2+-handling, ion channel, and cardiac-specific proteins in hvCOC compared to lower-order 2D and 3D cultures of the same constituent cells. Clinically-important, physiologically complex contractile parameters such as ejection fraction, developed pressure, and stroke work, as well as electrophysiological properties including action potential and conduction velocity were measured: hvCOC displayed key molecular and physiological characteristics of the native ventricle, and showed expected mechanical and electrophysiological responses to a range of pharmacological interventions (including positive and negative inotropes). We conclude that such "human-heart-in-a-jar" technology could facilitate the drug discovery process by providing human-specific preclinical data during early stage drug development.

Polycystin-2 Plays an Essential Role in Glucose Starvation-Induced Autophagy in Human Embryonic Stem Cell-Derived Cardiomyocytes.

Autophagy is a process essential for cell survival under stress condition. The patients with autosomal dominant polycystic kidney disease, which is caused by polycystin-1 or polycystin-2 (PKD2) mutation, display cardiovascular abnormalities and dysregulation in autophagy. However, it is unclear whether PKD2 plays a role in autophagy. In the present study, we explored the functional role of PKD2 in autophagy and apoptosis in human embryonic stem cell-derived cardiomyocytes. HES2 hESC line-derived cardiomyocytes (HES2-CMs) were transduced with adenoviral-based PKD2-shRNAs (Ad-PKD2-shRNAs), and then cultured with normal or glucose-free medium for 3 hours. Autophagy was upregulated in HES2-CMs under glucose starvation, as indicated by increased microtubule-associated protein 1 light chain 3-II level in immunoblots and increased autophagosome and autolysosome formation. Knockdown of PKD2 reduced the autophagic flux and increased apoptosis under glucose starvation. In Ca2+ measurement, Ad-PKD2-shRNAs reduced caffeine-induced cytosolic Ca2+ rise. Co-immunoprecipitation and in situ proximity ligation assay demonstrated an increased physical interaction of PKD2 with ryanodine receptor 2 (RyR2) under glucose starvation condition. Furthermore, Ad-PKD2-shRNAs substantially attenuated the starvation-induced activation of AMP-activated protein kinase (AMPK) and inactivation of mammalian target of rapamycin (mTOR). The present study for the first time demonstrates that PKD2 functions to promote autophagy under glucose starvation, thereby protects cardiomyocytes from apoptotic cell death. The mechanism may involve PKD2 interaction with RyR2 to alter Ca2+ release from sarcoplasmic reticulum, consequently modulating the activity of AMPK and mTOR, resulting in alteration of autophagy and apoptosis. Stem Cells 2018;36:501-513.