Wendy Keung

A simple, cost-effective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells.

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.

Aptamer-Based Microfluidic Electrochemical Biosensor for Monitoring Cell-Secreted Trace Cardiac Biomarkers

Continual monitoring of secreted biomarkers from organ-on-a-chip models is desired to understand their responses to drug exposure in a noninvasive manner. To achieve this goal, analytical methods capable of monitoring trace amounts of secreted biomarkers are of particular interest. However, a majority of existing biosensing techniques suffer from limited sensitivity, selectivity, stability, and require large working volumes, especially when cell culture medium is involved, which usually contains a plethora of nonspecific binding proteins and interfering compounds. Hence, novel analytical platforms are needed to provide noninvasive, accurate information on the status of organoids at low working volumes. Here, we report a novel microfluidic aptamer-based electrochemical biosensing platform for monitoring damage to cardiac organoids. The system is scalable, low-cost, and compatible with microfluidic platforms easing its integration with microfluidic bioreactors. To create the creatine kinase (CK)-MB biosensor, the microelectrode was functionalized with aptamers that are specific to CK-MB biomarker secreted from a damaged cardiac tissue. Compared to antibody-based sensors, the proposed aptamer-based system was highly sensitive, selective, and stable. The performance of the sensors was assessed using a heart-on-a-chip system constructed from human embryonic stem cell-derived cardiomyocytes following exposure to a cardiotoxic drug, doxorubicin. The aptamer-based biosensor was capable of measuring trace amounts of CK-MB secreted by the cardiac organoids upon drug treatments in a dose-dependent manner, which was in agreement with the beating behavior and cell viability analyses. We believe that, our microfluidic electrochemical biosensor using aptamer-based capture mechanism will find widespread applications in integration with organ-on-a-chip platforms for in situ detection of biomarkers at low abundance and high sensitivity.

Effect of engineered anisotropy on the susceptibility of human pluripotent stem cell-derived ventricular cardiomyocytes to arrhythmias.

Human (h) pluripotent stem cells (PSC) such as embryonic stem cells (ESC) can be directed into cardiomyocytes (CMs), representing a potential unlimited cell source for disease modeling, cardiotoxicity screening and myocardial repair. Although the electrophysiology of single hESC-CMs is now better defined, their multi-cellular arrhythmogenicity has not been thoroughly assessed due to the lack of a suitable experimental platform. Indeed, the generation of ventricular (V) fibrillation requires single-cell triggers as well as sustained multi-cellular reentrant events. Although native VCMs are aligned in a highly organized fashion such that electrical conduction is anisotropic for coordinated contractions, hESC-derived CM (hESC-CM) clusters are heterogenous and randomly organized, and therefore not representative of native conditions. Here, we reported that engineered alignment of hESC-VCMs on biomimetic grooves uniquely led to physiologically relevant responses. Aligned but not isotropic control preparations showed distinct longitudinal (L) and transverse (T) conduction velocities (CV), resembling the native human V anisotropic ratio (ARxa0=xa0LCV/TCVxa0=xa01.8-2.0). Importantly, the total incidence of spontaneous and inducible arrhythmias significantly reduced from 57% in controls to 17-23% of aligned preparations, thereby providing a physiological baseline for assessing arrhythmogenicity. As such, promotion of pro-arrhythmic effect (e.g., spatial dispersion by β adrenergic stimulation) could be better predicted. Mechanistically, such anisotropy-induced electrical stability was not due to maturation of the cellular properties of hESC-VCMs but their physical arrangement. In conclusion, not only do functional anisotropic hESC-VCMs engineered by multi-scale topography represent a more accurate model for efficacious drug discovery and development as well as arrhythmogenicity screening (of pharmacological and genetic factors), but our approach may also lead to future transplantable prototypes with improved efficacy and safety against arrhythmias.

Developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties of human pluripotent stem cell-derived cardiomyocytes

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are abundant sources of cardiomyocytes (CMs) for cell replacement therapy and other applications such as disease modeling, drug discovery and cardiotoxicity screening. However, hPSC-derived CMs display immature structural, electrophysiological, calcium-handling and metabolic properties. Here, we review various biological as well as physical and topographical cues that are known to associate with the development of native CMs in vivo to gain insights into the development of strategies for facilitated maturation of hPSC-CMs.

Acute impairment of contractile responses by 17β-estradiol is cAMP and protein kinase G dependent in vascular smooth muscle cells of the porcine coronary arteries

1 The aim of the present study was to investigate the involvement of adenosine 3′,5′‐cyclic monophosphate (cAMP) cascade in the acute impairment of contraction by 17β‐estradiol in porcine coronary arteries, and to elucidate the signaling pathway leading to the activation of this cascade by the hormone. 2 Isometric tension was recorded in isolated rings of porcine coronary arteries. 3 The contraction to U46619 was reduced significantly following 30u2003min incubation with 1u2003nM 17β‐estradiol or 1u2003nM isoproterenol. There was no additive effect when 17β‐estradiol and isoproterenol were administered together. The effect of 17β‐estradiol was mimicked by both the cyclic AMP analogue 8‐Br‐cAMP and the guanosine 3′,5′‐cyclic monophosphate (cyclic GMP) analogue 8‐Br‐cGMP. 4 In rings with and without endothelium, the modulatory effect of 17β‐estradiol was abolished by the adenylyl cyclase inhibitor, SQ 22536, but was unaffected by the guanylyl cyclase inhibitor, ODQ. 5 Both the cAMP antagonist Rp‐8‐Br‐cAMPS and the cGMP antagonist inhibitor Rp‐8‐Br‐cGMPS inhibited the effect of 17β‐estradiol. 6 The effect of 17β‐estradiol was unaffected by the protein kinase A inhibitor, KT5720, but was abolished by the protein kinase G (PKG) inhibitor, KT5823, which also abolished the effect of isoproterenol. 7 These data support our earlier findings that 17β‐estradiol (1u2003nM) acutely impairs contractile responses of porcine coronary arteries in vitro. This acute effect of 17β‐estradiol involves cAMP in vascular smooth muscles and the activation of PKG.

Non-genomic activation of adenylyl cyclase and protein kinase G by 17β-estradiol in vascular smooth muscle of the rat superior mesenteric artery

The aim of the present study was to investigate the signaling mechanisms underlying the non-genomic effects of estrogen in rat superior mesenteric arteries. Isometric tension was recorded in rings with or without endothelium. Changes in cyclic nucleotide levels and protein kinase (PK) activities were measured. Localization of estrogen receptors (ER) and caveolin-1 were visualized by confocal microscopy. 17β-Estradiol elicited a concentration-dependent relaxation. The relaxation was reduced by SQ 22536 (adenylyl cyclase inhibitor) and KT 5823 (PKG inhibitor) while ODQ (guanylyl cyclase inhibitor) and KT 5720 (PKA inhibitor) had no effect. At the physiological concentration of 1 nM, 17β-estradiol had no significant effect on relaxation but enhanced the relaxation to sodium nitroprusside. The enhancement of relaxation by 17β-estradiol was blocked by SQ 22536 and KT 5823. Although 1 nM 17β-estradiol or 10 nM sodium nitroprusside given alone had minimal effects on PKG activity, in their combined presence, a significant increase in PKG activity was observed. Confocal microscopy demonstrated that ERα and ERβ colocalized with caveolin-1 and PKG in vascular smooth muscle cells. The present findings suggest that 17β-estradiol enhances relaxation of vascular smooth muscle of the rat superior mesenteric artery by activating adenylyl cyclase, leading to an increase in cAMP which cross activates PKG in the caveolae. No detectable increase in total cAMP level was detected as these changes occurred in the caveolae. These results are consistent with the notion that 17β-estradiol mediates its effect in the distinct microdomains of the caveolae of the plasma membrane with colocalization of adenylyl cyclase and PKG.

NON-GENOMIC VASCULAR ACTIONS OF FEMALE SEX HORMONES: PHYSIOLOGICAL IMPLICATIONS AND SIGNALLING PATHWAYS

1 Epidemiological studies indicate a lower incidence of coronary heart disease in premenopausal women compared with age‐matched men and post‐menopausal women. Accumulating evidence suggests that this cardiovascular protection observed in premenopausal women is at least partially attributed to the direct action of oestrogens on the vascular system. 2 Research focused on vascular actions of 17β‐oestradiol indicates that this female sex hormone favourably modulates vascular reactivity at physiological concentrations. The vascular actions of 17β‐oestradiol appear independent of its genomic actions. Both endothelium‐dependent and ‐independent signalling cascades have been implicated in the vascular effects of 17β‐oestradiol. 3 However, clinical trials on hormone‐replacement therapy argue against a role of oestrogens in preventing the development of coronary heart disease. Supplementation with oestrogen is also complicated with the increased risk of breast and endometrial cancer. Hence, a better understanding of the vascular actions of 17β‐oestradiol will serve to enhance our understanding of its role in coronary heart disease.

Transcriptome-Guided Functional Analyses Reveal Novel Biological Properties and Regulatory Hierarchy of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes Crucial for Maturation

Abstract Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or non-chamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed IKATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Epigenetic regulation of the electrophysiological phenotype of human embryonic stem cell-derived ventricular cardiomyocytes: insights for driven maturation and hypertrophic growth.

Epigenetic regulation is implicated in embryonic development and the control of gene expression in a cell-specific manner. However, little is known about the role of histone methylation changes on human cardiac differentiation and maturation. Using human embryonic stem cells (hESCs) and their derived ventricular (V) cardiomyocytes (CMs) as a model, we examined trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) on promoters of genes associated with cardiac electrophysiology, contraction, and Ca(2+) handling. To avoid ambiguities due to heterogeneous chamber-specific types, hESC-derived ventricular cardiomyocytes (VCMs) were selected by dual zeocin-GFP expression under the transcriptional control of the MLC2v promoter and confirmed electrophysiologically by its signature action potential phenotype. High levels of H3K4me3 are present on pluripotency genes in hESCs with an absence of H3K27me3. Human ESC-VCMS, relative to hESCs, were characterized by a profound loss of H3K27me3 and an enrichment of H3K4me3 marks on cardiac-specific genes, including MYH6, MYH7, MYL2, cTNT, and ANF. Gene transcripts encoding key voltage-gated ion channels and Ca(2+)-handling proteins in hESC-VCMs were significantly increased, which could be attributed to a distinct pattern of differential H3K4me3 and H3K27me3 profiles. Treatment of hESC-VCMs with the histone deacetylase inhibitor valproic acid increased H3K4me3 on gene promoters, induced hypertrophic growth (as gauged by cell volume and capacitance), and augmented cardiac gene expression, but it did not affect electrophysiological properties of these cells. Hence, cardiac differentiation of hESCs involves a dynamic shift in histone methylation, which differentially affects VCM gene expression and function. We conclude that the epigenetic state of hESC-VCMs is dynamic and primed to promote growth and developmental maturation, but that proper environmental stimuli with chromatin remodeling will be required to synergistically trigger global CM maturation to a more adult-like phenotype.

Nongenomic responses to 17β-estradiol in male rat mesenteric arteries abolish intrinsic gender differences in vascular responses

The aim of the present study was to investigate the gender differences in the acute effects of 17β‐estradiol on the rat superior mesenteric artery. Isometric tension was measured in rings of mesenteric arteries from both male and female Sprague–Dawley rats. Relaxation to acetylcholine was not significantly different between arteries (with endothelium) from male and female rats in the absence or presence of 17β‐estradiol. After blockade of endothelium‐dependent hyperpolarizations with apamin (0.3u2003μM) plus charybdotoxin (0.1u2003μM), acute exposure to 17β‐estradiol (1u2003nM) for 30u2003min resulted in enhancement of relaxation to acetylcholine in arteries from male but not female rats. After acute exposure to 17β‐estradiol, mesenteric arteries from male rats were more sensitive to sodium nitroprusside than arteries from female rats. Contractions of mesenteric arteries to phenylephrine and 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (U46619) were greater in arteries from male rats than female rats. This difference was not detected after acute exposure to 17β‐estradiol. In preparations without endothelium, the enhancement of relaxation and reduction in contraction in arteries from male rats were preserved. These results suggest that there exists a gender difference in the response to the acute nongenomic modulatory effect of 17β‐estradiol in rat mesenteric arteries. Arteries from male rats seem to be more sensitive to the modulatory effects of 17β‐estradiol than arteries from female rats. The effect appears to be mainly at the level of the vascular smooth muscles.