Analysis of the Neurogenic Potential of a Three-Dimensional Culture of Mouse Dorsal Root Ganglia Explants

Background: Cultivation of dorsal root ganglia (DRGs) is a widely used approach for modeling the physiological and pathological conditions of the peripheral nervous system in vitro. Accumulating evidence indicates that postnatal DRGs harbor a pool of glial stem cells that are capable of differentiating into neurons following injury. Hence, extracted DRGs serving the stem cell pool may possess neurogenic potential ex vivo. AIM: In this study, we assessed the neurogenic potential of isolated DRGs and established a 3-dimensional organotypic culture model prepared from these DRGs. MATERIALS AND METHODS: Mouse-derived DRGs were short- and long-term cultivated in a three-dimensional gel. The morphology, ultrastructure, and gene expression profile of cultured DRGs were analyzed using light and electron microscopy, immunofluorescence staining, and RT-qPCR. RESULTS: We have demonstrated that the DRGs cultivated in Geltrex for 14 days stably generated a 3-dimensional periganglionic network consisting of radially-branched elongated cells. Immunofluorescence staining with antibodies against Tuj1 (neuron specific class III beta-tubulin) indicated that these observed cells were neuronal cells. Immunostaining with O4 antibodies confirmed that the Tuj1-marked cells were not migrating Schwann cells. Quantitative analysis of total RNA extracted from DRG culture by RT-qPCR revealed a significant increase in mRNA of key genes related to neurogenesis, such as Neurog1, Neurog2, and Sox2, indicating activation of the neurogenic program in DRG explants. Light and electron microscopy showed that adult sensory neurons were degenerated in the DRG explants and did not contribute to the periganglionic network. Using available confocal options, we estimated 3-dimensional parameters of the periganglionic network from 2-dimensional microscopy images to demonstrate the topography of Tuj1-expressing neuronal cells in a DRG cell culture. CONCLUSION: Thus, we have presented and visualized a novel physiologically relevant 3-dimensional culture model of DRG explants with neurogenic potential.