IADR Abstract Archives
P2X3 Expression and TRP Channel Modulation in an in-Vitro Human Sensory Neuronal Model Derived From Dental Pulp Stem Cells.
Objectives: Tissue inflammation, injury or stress can evoke ATP release from various cell sources. Accumulated extracellular ATP acts as an alarmin. The P2X3 is a membrane bound receptor gated by ATP. The transient receptor potential (TRP) channel family of cation channels detect mechanical and thermal stimuli as well as many endogenous and exogenous agonists. TRP channels serve as nociceptors responding to harmful stimuli on pulpal sensory nerves. The objective of this study was to investigate functional P2X3 expression in a model of peripheral neuronal equivalents (PNEs), derived from human dental pulp stem cells (hDPSCs). In addition, we sought to determine if ATP may induce TRP channel hyper-responsiveness through P2X3 activation in this model.
Methods: PNEs were differentiated from fibronectin-enriched hDPSCs isolated from dental pulp of extracted third molar teeth (Clarke et al, 2017). Characterisation of the neuronal phenotype of PNEs was carried out using immunocytochemistry (IHC) for neural markers, FluoVolt membrane depolarisation assay and single cell transcriptomic analysis. IHC was used to determine expression of P2X3 and TRP channels in PNEs. Confocal microfluorimetry was used to assess PNE responses to ATP ± P2X3 antagonist, AF-353. Confocal microfluorimetry was used to assess the effects of ATP and AF-353 on TRP channel responses.
Results: PNEs expressed the neuronal marker MAP2 and lost expression of fibroblastic marker FSP observed in hDPSC. hDPSC, neural progenitor and mature neuronal cell markers were observed in the transcriptome of PNEs; whilst no expression of odontoblast-like cell markers were observed by single cell transcriptomic analysis. P2X3, TRPA1 and TRPV4 protein was observed in PNEs by IHC. Functional assays showed ATP responses in PNEs were blocked by the P2X3 antagonist AF-353. TRPV4, but not TRPA1 responses became hyper-responsive with ATP incubation. The heightened TRPV4 responses in the presence of AF-353 could be prevented by AF-353 pre-incubation.
Conclusions: Functional P2X3 receptors are expressed on PNEs derived from hDPSCs. Hyper-responsiveness of TRPV4 was shown to be mediated by P2X3. This model has provided unique data on a role for alarmins in mediating sensory neuronal responses relevant to the dental pulp.
Methods: PNEs were differentiated from fibronectin-enriched hDPSCs isolated from dental pulp of extracted third molar teeth (Clarke et al, 2017). Characterisation of the neuronal phenotype of PNEs was carried out using immunocytochemistry (IHC) for neural markers, FluoVolt membrane depolarisation assay and single cell transcriptomic analysis. IHC was used to determine expression of P2X3 and TRP channels in PNEs. Confocal microfluorimetry was used to assess PNE responses to ATP ± P2X3 antagonist, AF-353. Confocal microfluorimetry was used to assess the effects of ATP and AF-353 on TRP channel responses.
Results: PNEs expressed the neuronal marker MAP2 and lost expression of fibroblastic marker FSP observed in hDPSC. hDPSC, neural progenitor and mature neuronal cell markers were observed in the transcriptome of PNEs; whilst no expression of odontoblast-like cell markers were observed by single cell transcriptomic analysis. P2X3, TRPA1 and TRPV4 protein was observed in PNEs by IHC. Functional assays showed ATP responses in PNEs were blocked by the P2X3 antagonist AF-353. TRPV4, but not TRPA1 responses became hyper-responsive with ATP incubation. The heightened TRPV4 responses in the presence of AF-353 could be prevented by AF-353 pre-incubation.
Conclusions: Functional P2X3 receptors are expressed on PNEs derived from hDPSCs. Hyper-responsiveness of TRPV4 was shown to be mediated by P2X3. This model has provided unique data on a role for alarmins in mediating sensory neuronal responses relevant to the dental pulp.