Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors

Abstract

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the CaV2 type, calcium permeable TRPV1 channels, and ionotropic GABAA receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca2+ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons

“…However, the functional excitability profile of the generated neurons must be investigated to provide a reliable functional baseline for the model's applicability in the investigation of diseases or the effect of drugs on defined ion channels. Functional profiling of hPSC-derived sensory neurons has primarily been focused on protocols that generate nociceptors (Chambers et al, 2012;Young et al, 2014;Blanchard et al, 2015;Eberhardt et al, 2015;Wainger et al, 2015;McDermott et al, 2019;Meents et al, 2019;Schoepf et al, 2020), rather than a heterogeneous population of sensory neurons akin to a more physiologically relevant model in which diverse populations of sensory neurons coexist. In this study, we sought to profile the spontaneously resulting mixed population of DRG sensory neurons at the transcriptional and functional levels including a detailed description of NGN2 iSN excitability patterns and their underlying voltage-dependent conductances.…”

Current EU regulatory requirements for the assessment of chemicals and cosmetic products: challenges and opportunities for introducing new approach methodologies

“…In vitro methods may also allow elucidating how inter-species differences can have an impact on chemical response, as shown for instance in Baumann et al study, where differences in chemical effects on neurodevelopmental key events were described comparing human and rat neurospheres (Baumann et al 2016 ). Several studies have highlighted species-specific differences, e.g., in the pace of development (Rayon et al 2020 ), in liver cytochrome P450 and transport protein (Hammer et al 2021 ), in the metabolic capacity and clearance of liver microsomes (Ma et al 2017 ), in the expression of GABA-A receptor in T lymphocytes (Mendu et al 2012 ), in the expression of nociceptive markers and ion channels between human and mouse iPSC-derived nociceptors (Schoepf et al 2020 ). Altogether, this underlines the importance to test chemical effects on human toxicological endpoints using human-relevant test systems.…”

NOCICEPTRA: Gene and microRNA signatures and their trajectories characterizing human iPSC-derived nociceptor maturation

“…Although native primary sensory afferents do not form autapses (92), iDNs express synaptic proteins (18). This is surprising but may be related to the unique feature of nociceptors to release neuropeptides and glutamate from differing vesicle pools at peripheral and spinal terminals (93).…”

“…Human-cell-based model systems, such as induced pluripotent stem cells (iPSCs), which allows directed cell-type specific programming into virtually any cell type by applying specific differentiation protocols (11)(12)(13)(14)(15), offers the opportunity to overcome this apparent translational paresis. Human iPSCs differentiated into nociceptor like cells express fundamental nociceptor-like characteristics and emerge as a valuable tool to explore mechanisms underlying hereditary pain disorders, such as erythromelalgia or migraine, and even to develop and select personalized treatments (16)(17)(18)(19)(20)(21)(22).…”

“…iPSC-derived sensory neuron (iDN) differentiationAD2, AD3 and 840 iPSCs were cultured on Matrigel (Corning) coated 6-well plates and maintained in mTeSR1 medium (STEMCELL Technologies). Nociceptor differentiation was performed as previously published(18,19). In brief, approximately 80 x 10 4 cells per 6-well plate were seeded as single-cells at passage 2 after thawing.…”