Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.

CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.

Anthracycline Toxicity: Light at the End of the Tunnel?

Anthracycline-induced cardiotoxicity (AIC) is a serious and common side effect of anthracycline therapy. Identification of genes and genetic variants associated with AIC risk has clinical potential as a cardiotoxicity predictive tool and to allow the development of personalized therapies. In this review, we provide an overview of the function of known AIC genes identified by association studies and categorize them based on their mechanistic implication in AIC. We also discuss the importance of functional validation of AIC-associated variants in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to advance the implementation of genetic predictive biomarkers. Finally, we review how patient-specific hiPSC-CMs can be used to identify novel patient-relevant functional targets and for the discovery of cardioprotectant drugs to prevent AIC. Implementation of functional validation and use of hiPSC-CMs for drug discovery will identify the next generation of highly effective and personalized cardioprotectants and accelerate the inclusion of approved AIC biomarkers into clinical practice.

TBXT dose sensitivity and the decoupling of nascent mesoderm specification from EMT progression in 2D human gastruloids.

In the nascent mesoderm, TBXT expression must be precisely regulated to ensure that cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell models of gastrulation and mesoderm differentiation. Multi-omic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprising wild-type, TBXT heterozygous or TBXT null human induced pluripotent stem cells reveal that varying TBXT dosage does not compromise the ability of a cell to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial-to-mesenchymal transition with wild type transitioning first, followed by TBXT heterozygous and then TBXT null. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that epithelial-to-mesenchymal transition progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.

Novel human iPSC models of neuroinflammation in neurodegenerative disease and regenerative medicine.

The importance of neuroinflammation in neurodegenerative diseases is becoming increasingly evident, and, in parallel, human induced pluripotent stem cell (hiPSC) models of physiology and pathology are emerging. Here, we review new advancements in the differentiation of hiPSCs into glial, neural, and blood-brain barrier (BBB) cell types, and the integration of these cells into complex organoids and chimeras. These advancements are relevant for modeling neuroinflammation in the context of prevalent neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). With awareness of current limitations, recent progress in the development and application of various hiPSC-derived models shows potential for aiding the identification of candidate therapeutic targets and immunotherapy approaches.

Maturing differentiated human pluripotent stem cells in vitro: methods and challenges.

Human pluripotent stem cells (hPSCs), derived from individuals or genetically modified with disease-related mutations and variants, have revolutionised studies of human disease. Researchers are beginning to exploit the extraordinary potential of stem cell technology to screen for new drugs to treat intractable diseases, ideally without side-effects. However, a major problem is that the differentiated cell types on which these models are based are immature; they resemble fetal and not adult cells. Here, we discuss the nature and hurdles of hPSC maturation, using cardiomyocytes as an example. We review methods used to induce cardiomyocyte maturation in culture and consider remaining challenges for their integration into research on human disease and drug development pipelines.

Harnessing developmental cues for cardiomyocyte production.

Developmental research has attempted to untangle the exact signals that control heart growth and size, with knockout studies in mice identifying pivotal roles for Wnt and Hippo signaling during embryonic and fetal heart growth. Despite this improved understanding, no clinically relevant therapies are yet available to compensate for the loss of functional adult myocardium and the absence of mature cardiomyocyte renewal that underlies cardiomyopathies of multiple origins. It remains of great interest to understand which mechanisms are responsible for the decline in proliferation in adult hearts and to elucidate new strategies for the stimulation of cardiac regeneration. Multiple signaling pathways have been identified that regulate the proliferation of cardiomyocytes in the embryonic heart and appear to be upregulated in postnatal injured hearts. In this Review, we highlight the interaction of signaling pathways in heart development and discuss how this knowledge has been translated into current technologies for cardiomyocyte production.

Retinal organoids from human-induced pluripotent stem cells: From studying retinal dystrophies to early diagnosis of Alzheimer's and Parkinson's disease.

Human-induced pluripotent stem cells (hiPSCs) have provided new methods to study neurodegenerative diseases. In addition to their wide application in neuronal disorders, hiPSCs technology can also encompass specific conditions, such as inherited retinal dystrophies. The possibility of evaluating alterations related to retinal disorders in 3D organoids increases the truthfulness of in vitro models. Moreover, both Alzheimer's (AD) and Parkinson's disease (PD) have been described as causing early retinal alterations, generating beta-amyloid protein accumulation, or affecting dopaminergic amacrine cells. This review addresses recent advances and future perspectives obtained from in vitro modeling of retinal diseases, focusing on retinitis pigmentosa (RP). Additionally, we depicted the possibility of evaluating changes related to AD and PD in retinal organoids obtained from potential patients long before the onset of the disease, constituting a valuable tool in early diagnosis. With this, we pointed out prospects in the study of retinal dystrophies and early diagnosis of AD and PD.

The impact of antidepressants on human neurodevelopment: Brain organoids as experimental tools.

The use of antidepressants during pregnancy benefits the mother's well-being, but the effects of such substances on neurodevelopment remain poorly understood. Moreover, the consequences of early exposure to antidepressants may not be immediately apparent at birth. In utero exposure to selective serotonin reuptake inhibitors (SSRIs) has been related to developmental abnormalities, including a reduced white matter volume. Several reports have observed an increased incidence of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) after prenatal exposure to SSRIs such as sertraline, the most widely prescribed SSRI. The advent of human-induced pluripotent stem cell (hiPSC) methods and assays now offers appropriate tools to test the consequences of such compounds for neurodevelopment in vitro. In particular, hiPSCs can be used to generate cerebral organoids - self-organized structures that recapitulate the morphology and complex physiology of the developing human brain, overcoming the limitations found in 2D cell culture and experimental animal models for testing drug efficacy and side effects. For example, single-cell RNA sequencing (scRNA-seq) and electrophysiological measurements on organoids can be used to evaluate the impact of antidepressants on the transcriptome and neuronal activity signatures in developing neurons. While the analysis of large-scale transcriptomic data depends on dimensionality reduction methods, electrophysiological recordings rely on temporal data series to discriminate statistical characteristics of neuronal activity, allowing for the rigorous analysis of the effects of antidepressants and other molecules that affect the developing nervous system, especially when applied in combination with relevant human cellular models such as brain organoids.

In vitro modeling of cranial placode differentiation: Recent advances, challenges, and perspectives.

Cranial placodes are transient ectodermal thickenings that contribute to a diverse array of organs in the vertebrate head. They develop from a common territory, the pre-placodal region that over time segregates along the antero-posterior axis into individual placodal domains: the adenohypophyseal, olfactory, lens, trigeminal, otic, and epibranchial placodes. These placodes terminally differentiate into the anterior pituitary, the lens, and contribute to sensory organs including the olfactory epithelium, and inner ear, as well as several cranial ganglia. To study cranial placodes and their derivatives and generate cells for therapeutic purposes, several groups have turned to in vitro derivation of placodal cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). In this review, we summarize the signaling cues and mechanisms involved in cranial placode induction, specification, and differentiation in vivo, and discuss how this knowledge has informed protocols to derive cranial placodes in vitro. We also discuss the benefits and limitations of these protocols, and the potential of in vitro cranial placode modeling in regenerative medicine to treat cranial placode-related pathologies.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation [mean ± SD]: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons.

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function.

Laminin 511 and WNT signalling sustain prolonged expansion of hiPSC-derived hippocampal progenitors.

Using the timely re-activation of WNT signalling in neuralizing human induced pluripotent stem cells (hiPSCs), we have produced neural progenitor cells with a gene expression profile typical of human embryonic dentate gyrus (DG) cells. Notably, in addition to continuous WNT signalling, a specific laminin isoform is crucial to prolonging the neural stem state and to extending progenitor cell proliferation for over 200 days in vitro. Laminin 511 is indeed specifically required to support proliferation and to inhibit differentiation of hippocampal progenitor cells for extended time periods when compared with a number of different laminin isoforms assayed. Global gene expression profiles of these cells suggest that a niche of laminin 511 and WNT signalling is sufficient to maintain their capability to undergo typical hippocampal neurogenesis. Moreover, laminin 511 signalling sustains the expression of a set of genes responsible for the maintenance of a hippocampal neurogenic niche. Finally, xenograft of human DG progenitors into the DG of adult immunosuppressed host mice produces efficient integration of neurons that innervate CA3 layer cells spanning the same area of endogenous hippocampal neuron synapses.

Endothelin-1 influences mechanical properties and contractility of hiPSC derived cardiomyocytes resulting in diastolic dysfunction.

A better understanding of the underlying pathomechanisms of diastolic dysfunction is crucial for the development of targeted therapeutic options with the aim to increase the patients' quality of life. In order to shed light on the processes involved, suitable models are required. Here, effects of endothelin-1 (ET-1) treatment on cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were investigated. While it is well established, that ET-1 treatment induces hypertrophy in cardiomyocytes, resulting changes in cell mechanics and contractile behavior with focus on relaxation have not been examined before. Cardiomyocytes were treated with 10 nM of ET-1 for 24 h and 48 h, respectively. Hypertrophy was confirmed by real-time deformability cytometry (RT-DC) which was also used to assess the mechanical properties of cardiomyocytes. For investigation of the contractile behavior, 24 h phase contrast video microscopy was applied. To get a deeper insight into changes on the molecular biological level, gene expression analysis was performed using the NanoString nCounter® cardiovascular disease panel. Besides an increased cell size, ET-1 treated cardiomyocytes are stiffer and show an impaired relaxation. Gene expression patterns in ET-1 treated hiPSC derived cardiomyocytes showed that pathways associated with cardiovascular diseases, cardiac hypertrophy and extracellular matrix were upregulated while those associated with fatty acid metabolism were downregulated. We conclude that alterations in cardiomyocytes after ET-1 treatment go far beyond hypertrophy and represent a useful model for diastolic dysfunction.

Adipocyte-mediated electrophysiological remodeling of human stem cell - derived cardiomyocytes.

Adipocytes normally accumulate in the epicardial and pericardial layers around the human heart, but their infiltration into the myocardium can be proarrhythmic. METHODS AND RESULTS: Human adipose derived stem/stromal cells and human induced pluripotent stem cells (hiPSC) were differentiated, respectively into predominantly white fat-like adipocytes (hAdip) and ventricular cardiomyocytes (CMs). Adipocytes cultured in CM maintenance medium (CM medium) maintained their morphology, continued to express adipogenic markers, and retained clusters of intracellular lipid droplets. In contrast, hiPSC-CMs cultivated in adipogenic growth medium displayed abnormal cell morphologies and more clustering across the monolayer. Pre-plated hiPSC-CMs co-cultured in direct contact with hAdips in CM medium displayed prolonged action potential durations, increased triangulation, slowed conduction velocity, increased conduction velocity heterogeneity, and prolonged calcium transients. When hAdip-conditioned medium was added to monolayer cultures of hiPSC-CMs, results similar to those recorded with direct co-cultures were observed. Both co-culture and conditioned medium experiments resulted in increases in transcript abundance of SCN10A, CACNA1C, SLC8A1, and RYR2, with a decrease in KCNJ2. Human adipokine immunoblots revealed the presence of cytokines that were elevated in adipocyte-conditioned medium, including MCP-1, IL-6, IL-8 and CFD that could induce electrophysiological changes in cultured hiPSC-CMs. CONCLUSIONS: Co-culture of hiPSC-CMs with hAdips reveals a potentially pathogenic role of infiltrating human adipocytes on myocardial tissue. In the absence of structural changes, hAdip paracrine release alone is sufficient to cause CM electrophysiological dysfunction mirroring the co-culture conditions. These effects, mediated largely by paracrine mechanisms, could promote arrhythmias in the heart.

Developmental Pb exposure increases AD risk via altered intracellular Ca2+ homeostasis in hiPSC-derived cortical neurons.

Exposure to environmental chemicals such as lead (Pb) during vulnerable developmental periods can result in adverse health outcomes later in life. Human cohort studies have demonstrated associations between developmental Pb exposure and Alzheimer's disease (AD) onset in later life which were further corroborated by findings from animal studies. The molecular pathway linking developmental Pb exposure and increased AD risk, however, remains elusive. In this work, we used human iPSC-derived cortical neurons as a model system to study the effects of Pb exposure on AD-like pathogenesis in human cortical neurons. We exposed neural progenitor cells derived from human iPSC to 0, 15, and 50 ppb Pb for 48 h, removed Pb-containing medium, and further differentiated them into cortical neurons. Immunofluorescence, Western blotting, RNA-sequencing, ELISA, and FRET reporter cell lines were used to determine changes in AD-like pathogenesis in differentiated cortical neurons. Exposing neural progenitor cells to low-dose Pb, mimicking a developmental exposure, can result in altered neurite morphology. Differentiated neurons exhibit altered calcium homeostasis, synaptic plasticity, and epigenetic landscape along with elevated AD-like pathogenesis markers, including phosphorylated tau, tau aggregates, and Aß42/40. Collectively, our findings provide an evidence base for Ca dysregulation caused by developmental Pb exposure as a plausible molecular mechanism accounting for increased AD risk in populations with developmental Pb exposure.

Cadherin-5 facilitated the differentiation of human induced pluripotent stem cells into sinoatrial node-like pacemaker cells by regulating ß-catenin.

Our study was conducted to investigate whether cadherin-5 (CDH5), a vascular endothelial cell adhesion glycoprotein, could facilitate the differentiation of human induced pluripotent stem cells (hiPSCs) into sinoatrial node-like pacemaker cells (SANLPCs), following previous findings of silk-fibroin hydrogel-induced direct conversion of quiescent cardiomyocytes into pacemaker cells in rats through the activation of CDH5. In this study, the differentiating hiPSCs were treated with CDH5 (40 ng/mL) between Day 5 and 7 during cardiomyocytes differentiation. The findings in the present study demonstrated that CDH5 stimulated the expression of pacemaker-specific markers while suppressing markers associated with working cardiomyocytes, resulting in an increased proportion of SANLPCs among hiPSCs-derived cardiomyocytes (hiPSC-CMs) population. Moreover, CDH5 induced typical electrophysiological characteristics resembling cardiac pacemaker cells in hiPSC-CMs. Further mechanistic investigations revealed that the enriched differentiation of hiPSCs into SANLPCs induced by CDH5 was partially reversed by iCRT14, an inhibitor of ß-catenin. Therefore, based on the aforementioned findings, it could be inferred that the regulation of ß-catenin by CDH5 played a crucial role in promoting the enriched differentiation of hiPSCs into SANLPCs, which presents a novel avenue for the construction of biological pacemakers in forthcoming research.

Decoding HiPSC-CM's Response to SARS-CoV-2: mapping the molecular landscape of cardiac injury.

BACKGROUND: Acute cardiac injury caused by coronavirus disease 2019 (COVID-19) increases mortality. Acute cardiac injury caused by COVID-19 requires understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly infects cardiomyocytes. This study provides a solid foundation for related studies by using a model of SARS-CoV-2 infection in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) at the transcriptome level, highlighting the relevance of this study to related studies. SARS-CoV-2 infection in hiPSC-CMs has previously been studied by bioinformatics without presenting the full molecular biological process. We present a unique bioinformatics view of the complete molecular biological process of SARS-CoV-2 infection in hiPSC-CMs. METHODS: To validate the RNA-seq datasets, we used GSE184715 and GSE150392 for the analytical studies, GSE193722 for validation at the cellular level, and GSE169241 for validation in heart tissue samples. GeneCards and MsigDB databases were used to find genes associated with the phenotype. In addition to differential expression analysis and principal component analysis (PCA), we also performed protein-protein interaction (PPI) analysis, functional enrichment analysis, hub gene analysis, upstream transcription factor prediction, and drug prediction. RESULTS: Differentially expressed genes (DEGs) were classified into four categories: cardiomyocyte cytoskeletal protein inhibition, proto-oncogene activation and inflammation, mitochondrial dysfunction, and intracellular cytoplasmic physiological function. Each of the hub genes showed good diagnostic prediction, which was well validated in other datasets. Inhibited biological functions included cardiomyocyte cytoskeletal proteins, adenosine triphosphate (ATP) synthesis and electron transport chain (ETC), glucose metabolism, amino acid metabolism, fatty acid metabolism, pyruvate metabolism, citric acid cycle, nucleic acid metabolism, replication, transcription, translation, ubiquitination, autophagy, and cellular transport. Proto-oncogenes, inflammation, nuclear factor-kappaB (NF-κB) pathways, and interferon signaling were activated, as well as inflammatory factors. Viral infection activates multiple pathways, including the interferon pathway, proto-oncogenes and mitochondrial oxidative stress, while inhibiting cardiomyocyte backbone proteins and energy metabolism. Infection limits intracellular synthesis and metabolism, as well as the raw materials for mitochondrial energy synthesis. Mitochondrial dysfunction and energy abnormalities are ultimately caused by proto-oncogene activation and SARS-CoV-2 infection. Activation of the interferon pathway, proto-oncogene up-regulation, and mitochondrial oxidative stress cause the inflammatory response and lead to diminished cardiomyocyte contraction. Replication, transcription, translation, ubiquitination, autophagy, and cellular transport are among the functions that decline physiologically. CONCLUSION: SARS-CoV-2 infection in hiPSC-CMs is fundamentally mediated via mitochondrial dysfunction. Therapeutic interventions targeting mitochondrial dysfunction may alleviate the cardiovascular complications associated with SARS-CoV-2 infection.

Dopamine­iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease.

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

The human-specific nicotinic receptor subunit CHRFAM7A reduces α7 receptor function in human induced pluripotent stem cells-derived and transgenic mouse neurons.

We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists.

High-throughput methods for cardiac cellular electrophysiology studies: the road to personalized medicine.

Personalized medicine refers to the tailored application of medical treatment at an individual level, considering the specific genotype or phenotype of each patient for targeted therapy. In the context of cardiovascular diseases, implementing personalized medicine is challenging due to the high costs involved and the slow pace of identifying the pathogenicity of genetic variants, deciphering molecular mechanisms of disease, and testing treatment approaches. Scalable cellular models such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as useful in vitro tools that reflect individual patient genetics and retain clinical phenotypes. High-throughput functional assessment of these constructs is necessary to rapidly assess cardiac pathogenicity and test new therapeutics if personalized medicine is to become a reality. High-throughput photometry recordings of single cells coupled with potentiometric probes offer cost-effective alternatives to traditional patch-clamp assessments of cardiomyocyte action potential characteristics. Importantly, automated patch-clamp (APC) is rapidly emerging in the pharmaceutical industry and academia as a powerful method to assess individual membrane-bound ionic currents and ion channel biophysics over multiple cells in parallel. Now amenable to primary cell and hiPSC-CM measurement, APC represents an exciting leap forward in the characterization of a multitude of molecular mechanisms that underlie clinical cardiac phenotypes. This review provides a summary of state-of-the-art high-throughput electrophysiological techniques to assess cardiac electrophysiology and an overview of recent works that successfully integrate these methods into basic science research that could potentially facilitate future implementation of personalized medicine at a clinical level.