191 Canine Mesenchymal Stem Cells as Therapeutic Agents in Spinal Cord Injured Dog

Abstract

In the recent era of veterinary research, stem cells have gained special attention due to their efficiency and use in clinical applications. Mesenchymal stem cells (MSC) have been extensively studied over decades, and their prospect for clinical application is recognised in human medicine. Despite numerous reports in veterinary clinical trials of stem cells, few studies have been presented regarding the in vitro characterisation of canine mesenchymal stem cells (cMSC). Therefore, their efficacy as therapeutic agents in vitro has not been much elucidated. Canine adipose-derived mesenchymal stem cells (cAMSC) were characterised as per International Society for Cellular Therapy guidelines. Culturing cells showed spindle-like morphology and high proliferation rate. They displayed positive expression of mesenchymal markers CD44, CD90, and CD105, and lacked expression of CD34 and CD45. They were also positive for expression of pluripotency-related transcription factors (Oct3/4, Nanog, and Sox2) and showed differentiation potential towards mesodermal lineages. The cAMSC were further analysed for the neuronal trans-differentiation potential. Under appropriate differentiation conditions, cAMSC displayed distinctive dendritic morphology along with axon projections. Neuronal specific genes including Nestin, β-tubulin, neurofilament protein (NF-M, NF-H), and nerve growth factor (NGF) were also positively expressed. Nevertheless, functional analysis of neuronal differentiated cAMSC displayed voltage dependence and kinetics for transient K⁺ and Na⁺ currents (Ito). Both K⁺ and Na⁺ currents were recorded in differentiated MSC by voltage steps (between −120 and +60 mV for K⁺ currents, −40 and +50 mV for Na⁺ currents), whereas control undifferentiated MSC lacked the currents. Taken together, we concluded that the cAMSC have potential to differentiate into neuron-like cells. Based on these findings, we transplanted cAMSC into the spinal cord injured dogs to evaluate their clinical efficiency under approved medical guidelines set by Gyeongsang National University Animal Medical Center (Korea). Neurological examination showed that the injured dog had undergone hind limb paralysis and lost deep pain sensation due to an L2 spinal cord lesion, as detected by CT and MRI. The dog was diagnosed with traumatic L2 intradural spinal cord contusion, and decompression surgery was performed, but deep pain sensation did not recover. Therefore, each cAMSC (diluted in 0.5 mL of saline) was transplanted into spinal cord segment (L2~L3) 5 times at 1-week intervals. The dog showed mild recovery of deep pain sensation by neurological examinations and exhibited gradual improvement in hind limb function. Finally, we concluded that transplantation of cAMSC has a beneficial therapeutic effect on spinal cord injury. This study also provides a significant advantage in understanding the potential of MSC-based products in veterinary clinical applications.