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.
Highlights
Over the past decades, animal experiments have been the gold standard for pain research and, without question, have tremendously contributed to the current knowledge in the field
Differentiation of human induced pluripotent stem cells (iPSCs) to iPSC-derived nociceptors (iDNs) was performed by dual SMAD inhibition, leading to neural crest-like cells followed by an overlapping inhibition of glycogen synthase kinase 3 (GSK3β), vascular endothelial growth factor (VEGF), and Notch signaling (3i inhibition), and long-term maintenance in neurobasal medium infused with the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), as firstly described by Chambers, Qi, Mica, Lee, Zhang, Niu, Bilsland, Cao, Stevens, Whiting, Shi and Studer [4]
Characterization of early stage iDNs and sensory neurons obtained from mature mouse dorsal root ganglia (DRG) was performed by quantification of BRN3A and ISL1 expression, which are two transcription factors with critical implications for sensory neuron development [32]
Summary
Animal experiments have been the gold standard for pain research and, without question, have tremendously contributed to the current knowledge in the field. Besides ethical concerns, the results achieved in rodent models are confounded by interspecies differences when translating findings to humans. Brain Sci. 2020, 10, 344 primary sensory neurons, are unattainable in a living human being and have even proven complicated to be obtained post mortem [1]. The caveat of species-to-species differences has become evident by recent studies that have compared specific nociceptive markers between human post-mortem and mouse neurons obtained from the DRG; the human proteome includes 286 proteins that are not even expressed in rodent DRG [2]. In patch-clamp experiments, iDNs express tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) voltage-gated sodium currents with unexpected differences to their counterparts from rodent DRG. Novel mechanistic insight into the development of rare congenital disorders with an inability to feel pain or extreme pain has emerged from patient-derived nociceptor studies [5,6,7,8,9,10,11,12]
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