Sensorineural hearing loss is a common disability found worldwide which is associated with a degeneration of spiral ganglion neurons (SGN). It is a challenge to restore SGN due to the permanent degeneration and viability of SGN is requisite for patients to receive an advantage from hearing aid devices. Human dental pulp stem cells (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) are self-renewing stem cells that originate from the neural crest during development. These stem cells have a high potential for neuronal differentiation. This is primarily due to their multilineage differentiation potential and their relative ease of access. Previously, we have shown the ability of these stem cell types to differentiate into spiral ganglion neuron-like cells. In this study, we induced the cells into neural precursor cells (NPC) and cocultured with auditory brainstem slice (ABS) encompassing cochlear nucleus by the Stoppini method. We also investigated their ability to differentiate after 2 weeks and 4 weeks in coculture. Neuronal differentiation of DPSC-NPC and SHED-NPC was higher expression of specific markers to SGN, TrkB, and Gata3, compared to monoculture. The cells also highly expressed synaptic vesicle protein (SV2A) and exhibited intracellular calcium oscillations. Our findings demonstrated the possibility of using DPSCs and SHEDs as an autologous stem cell-based therapy for sensorineural hearing loss patients. K E Y W O R D S auditory, brainstem slice (ABS), deciduous teeth (SHED), human dental pulp stem cell (DPSC), spiral ganglion neuron (SGN), stem cell from human exfoliated
Objectives: Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology: After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results: Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion: These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.
Objectives The purpose of the present study was to evaluate the efficacy of spiral ganglion neuron (SGN) regeneration after dental pulp stem cell (DPSC) transplantation in a rat sensorineural hearing loss (HL) model. Materials and Methods Sham or experimental HL was induced in adult Sprague–Dawley rats by cochlear round window surgery. An HL rat model was established with a single 10 mM ouabain intratympanic injection. After 7 days, the rats received DPSCs, stem cells from human exfoliated deciduous teeth (SHED), or culture medium in the sutural area to establish four groups: sham, HL-DPSC, HL-SHED, and HL-medium. Histological analyses were performed at 4, 7, and 10 weeks after transplantation, and the number of SGNs, specific SGN protein expression, and the function of SGNs were evaluated. Statistical Analysis Data were statistically by MS Excel and SPSS v.15.0. Intergroup level of significance was determined via a one-way analysis of variance and Duncan's multiple range test with 95% confidence intervals. Results New SGN formation was observed in the HL-DPSC and HL-SHED rat groups. The number of SGNs was significantly higher in the HL-DPSC and HL-SHED groups than in the HL-medium group over 4 to 10-week survival period. HL-DPSC rats exhibited higher SGN density compared with that in HL-SHED group, which was statistically significant at week 10. The regenerated SGNs expressed cochlear wiring regulator GATA-binding-protein 3. Moreover, the SGNs from the HL-DPSC group also exhibited a higher expression of synaptic vesicle protein and regulated action potential-dependent neurotransmitter release compared with SGNs from the HL-SHED group. Conclusions Our findings suggest that DPSCs and SHED repair and regenerate SGNs in rat HL model. Dental pulp stem cells represent a promising treatment strategy for restoring damage to the sensory circuits associated with deafness.
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