Peripheral sensory neurons are a primary effector in pain neurotransmission, and have become a useful cellular model for the study of pain. While rodent tissue has historically served as a source of these neurons, it has become increasingly clear that pain mechanisms in rodents and humans are substantially divergent. Sensory neurons harvested from cadaveric human tissue serve as a superior translational model for studying pain mechanisms, however their relative paucity limits their widespread utility. Theoretically, sensory neurons manufactured from human induced pluripotent stem cells (hiPSCs) could help bridge this translational gap given their relative abundance and potential similarity to primary human tissue. However, hiPSC-derived sensory neurons manufactured with the most common methodologies correlate poorly to human tissue both transcriptionally and functionally. In the present work, we compare a population of hiPSC-derived sensory neurons (hiSNs) generated using an accelerated directed differentiation method to previously published datasets and find this population to more closely resemble human primary dorsal root ganglia transcriptionally. Furthermore, we evaluate the heterogeneity of this novel population via single nucleus RNA sequencing and find it resembles specific nociceptor and mechanoreceptor subsets found in vivo. Finally, we assay the functionality of this population with high throughput automated patch clamp electrophysiology recordings of voltage-gated TTX-sensitive and TTX-resistant sodium (Nav), potassium channels (Kv), ligand-gated ionotropic GABA and P2X receptors, and action potentials. Overall, we find this population of hiSNs demonstrates measurable expression and functional activity across multiple pain-relevant targets, making it suitable for interrogating diverse pain mechanisms on a fully humanized platform.

EDITORIAL article Front. Mol. Neurosci., 31 May 2024Sec. Molecular Signalling and Pathways Volume 17 - 2024 | https://doi.org/10.3389/fnmol.2024.1414816

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.

IntroductionIn response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet.MethodsThe proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction.ResultsThe selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation).DiscussionThis work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.

Introduction: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. Methods: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and infra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. Results: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. Discussion: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Conventionally, manual patch-clamp electrophysiological approaches are the gold standard for studying ion channel function in neurons. However, these approaches are labor-intensive, yielding low-throughput results, and are therefore not amenable for compound profiling efforts during the early stages of drug discovery. The SyncroPatch 384PE has been successfully implemented for pharmacological experiments in heterologous overexpression systems that may not reproduce the function of voltage-gated ion channels in a native, heterogeneous environment. Here, we describe a protocol allowing the characterization of endogenous voltage-gated potassium (Kv) and sodium (Nav) channel function in developing primary rat cortical cultures, allowing investigations at a significantly improved throughput compared with manual approaches. Key neuronal marker expression and microelectrode array recordings of electrophysiological activity over time correlated well with neuronal maturation. Gene expression data revealed high molecular diversity in Kv and Nav subunit composition throughout development. Voltage-clamp experiments elicited three major current components composed of inward and outward conductances. Further pharmacological experiments confirmed the endogenous expression of functional Kv and Nav channels in primary cortical neurons. The major advantages of this approach compared with conventional manual patch-clamp systems include unprecedented improvements in experimental ease and throughput for ion channel research in primary neurons. These efforts demonstrated feasibility for primary neuronal ion channel investigation with the SyncroPatch, providing the foundation for future studies characterizing biophysical changes in endogenous ion channels in primary systems associated with disease or development.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Human induced pluripotent stem cells (hiPSCs) are relevant for cardiac safety testing due to their validated predictivity as described in recent publications derived from the comprehensive in vitro proarrhythmia assay (CiPA) study. We combined automated patch clamp (APC), impedance and extracellular field potential (EFP) measurements to study cardiac ion channels in cell lines and hiPSC-CMs. Cell lines expressing different cardiac ion channels were recorded on two different APC instruments (8 or 384 wells simultaneously) at room and physiological temperature, at 4 different sites. Within the myocyte validation study of the CiPA initiative, hiPSC-CMs from different providers were used on a device combining impedance-based contractility and extracellular field potential (EFP) recordings. Within the ion channels working group of the CiPA initiative, ion channel data on 7 cardiac ion channel currents were measured. The effects of >20 CiPA reference compounds deemed low, intermediate and high risk by the FDA were investigated. In parallel to APC investigations, arrhythmogenicity of this and other compounds were investigated via contractility and field potential recordings in 2D monolayers of iPSC-CM. Our results show that high risk compounds such as dofetilide prolong field potential duration which resulted in the detection of arrhythmic events in both impedance and EFP recordings. Datasets from multiple sites and cell types will be shown and compared. Cross-site/cell comparisons of APC ion channel data and myocyte repolarization data was combined in order to investigate the mode of action of reference compounds. This promotes a good understanding of drug-induced arrhythmia and thus represents a valuable approach in drug development efforts.