There has been considerable progress in identifying signaling pathways directing the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types including neurons. However, extrinsic factor-based differentiation of hPSCs is a slow, step-wise process mimicking the protracted timing of normal human development.
Using a small molecule screen we identified a combination of five small molecule pathway inhibitors sufficient to yield hPSC-derived neurons at >75% efficiency within 10 days of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors including TTX-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli including ATP and capsaicin. Neuronal fate acquisition occurs three-fold faster than during in vivo1 development suggesting that use of small molecule pathway inhibitors could develop into a general strategy for accelerating developmental timing in vitro. The quick and high efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain.
One sentence summary: Bench to bedside translation using iPSC to characterise phenotype and pharmacology in primary erythromelalgia, an inherited chronic pain condition.
ABSTRACTIn common with other chronic pain conditions, inherited erythromelalgia (IEM) represents a significant unmet medical need. The peripherally expressed SCN9A encoded sodium channel Nav1.7 plays a critical role in IEM with gain-of-function leading to aberrant sensory neuronal activity and extreme pain, particularly in response to heat. In five carefully phenotyped IEM patients, a novel highly potent and selective Nav1.7 blocker reduced heat-induced pain in the majority of subjects. In four of the five subjects we used induced pluripotent stem cell (iPSC) technology to create sensory neurons which uniquely emulated the clinical phenotype of hyperexcitability and aberrant responses to heat stimuli. When we compared the severity of the clinical phenotype with the iPSC-derived sensory neuron hyperexcitability we saw a trend towards a correlation for individual mutations. The in vitro IEM phenotype was sensitive to Nav1.7 blockers, including the clinical test agent. Given the importance of peripherally expressed sodium channels in many pain conditions, this translational approach is likely to have broader utility to a wide range of pain and sensory conditions. This emphasizes the use of iPSC approaches to bridge between clinical and preclinical studies, enabling greater understanding of a disease and the response to a therapeutic agent in defined patient populations.
The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.
The receptor tyrosine kinase product of the anaplastic lymphoma kinase (ALK) gene has been implicated in oncogenesis as a product of several chromosomal translocations, although its endogeneous role in the hematopoietic and neural systems has remained poorly understood. We describe that the generation of animals homozygous for a deletion of the ALK tyrosine kinase domain leads to alterations in adult brain function. Evaluation of adult ALK homozygotes (HOs) revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain. ALK HO animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel objectrecognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. Altogether, these results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia and depression, with an underlying deregulated monoaminergic signalling.
The neurotrophin family of growth factors, comprised of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), is implicated in the physiology of chronic pain. Given the clinical efficacy of anti-NGF monoclonal antibody (mAb) therapies, there is significant interest in the development of small molecule modulators of neurotrophin activity. Neurotrophins signal through the tropomyosin related kinase (Trk) family of tyrosine kinase receptors, hence Trk kinase inhibition represents a potentially "druggable" point of intervention. To deliver the safety profile required for chronic, nonlife threatening pain indications, highly kinase-selective Trk inhibitors with minimal brain availability are sought. Herein we describe how the use of SBDD, 2D QSAR models, and matched molecular pair data in compound design enabled the delivery of the highly potent, kinase-selective, and peripherally restricted clinical candidate PF-06273340.
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