Abstract
Several methods have been developed to generate neurons from other cell types for performing regeneration therapy and in vitro studies of central nerve disease. Small molecules (SMs) can efficiently induce neuronal features in human and rodent fibroblasts without transgenes. Although canines have been used as a spontaneous disease model of human central nerve, efficient neuronal reprogramming method of canine cells have not been well established. We aimed to induce neuronal features in adult canine dermal fibroblasts (ACDFs) by SMs and assess the permanency of these changes. ACDFs treated with eight SMs developed a round-shaped cell body with branching processes and expressed neuronal proteins, including βIII-tubulin, microtubule-associated protein 2 (MAP2), and neurofilament-medium. Transcriptome profiling revealed the upregulation of neuron-related genes, such as SNAP25 and GRIA4, and downregulation of fibroblast-related genes, such as COL12A1 and CCN5. Calcium fluorescent imaging demonstrated an increase in intracellular Ca2+ concentration upon stimulation with glutamate and KCl. Although neuronal features were induced similarly in basement membrane extract droplet culture, they diminished after culturing without SMs or in vivo transplantation into an injured spinal cord. In conclusion, SMs temporarily induce neuronal features in ACDFs. However, the analysis of bottlenecks in the neuronal induction is crucial for optimizing the process.