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 physiological and pathological conditions of the peripheral nervous system in vitro. Existing scientific data indicates that postnatal DRGs contain a pool of glial stem cells capable of differentiating into neurons after injury. Therefore, isolated DRGs containing a stem cell pool may retain neurogenic potential ex vivo. However, no studies in the available scientific data have investigated the preservation of the regenerative potential of DRGs ex vivo.

AIM: This study aimed to evaluate the neurogenic potential of isolated DRGs and establish a three-dimensional organotypic culture from these explants.

METHODS: DRGs were isolated from the cervical, thoracic, and lumbar regions of the spine and cultured short-term and long-term as three-dimensional explants in Geltrex gel. The morphology, ultrastructure, and gene expression profile of the cultured DRGs were analyzed using light and electron microscopy, immunofluorescent staining, and quantitative reverse transcription polymerase chain reaction.

RESULTS: DRGs cultured in Geltrex for 14 days consistently generated a three-dimensional periganglionic network composed of radially branching elongated cells. Immunofluorescent staining with Tuj1 antibodies (neuron-specific class III β-tubulin) demonstrated that the observed cells were neuronal. Immunostaining with O4 antibodies confirmed that Tuj1-positive cells were not migrating Schwann cells. Quantitative reverse transcription polymerase chain reaction analysis of total RNA extracted from the DRG explant cultures revealed a significant increase in mRNA levels of key neurogenesis-related genes, such as Neurog1, Neurog2, and Sox2, indicating activation of a neurogenic program in the cultured explants. Light and electron microscopy showed that adult sensory neurons degenerated within the DRG explants and did not contribute to the periganglionic network. Using available confocal capabilities, we evaluated three-dimensional parameters of the periganglionic network from two-dimensional microscopic images to demonstrate the topography of Tuj1-expressing neuronal cells in a DRG explant culture.

CONCLUSION: We characterized a three-dimensional culture of DRGs. Isolated explants demonstrate regenerative capacity under in vitro conditions. They may represent a novel organotypic model with neurogenic potential. The obtained model may be relevant for studying regenerative processes in DRGs, expanding research opportunities beyond traditional stem cell–derived models.