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1. were also performed. Olby scores in the ASC group increased from 2 weeks after transplantation and were significantly higher than C and V groups until 8 weeks (p< 0.05). However, there were no significant differences between the C and V groups. Nerve conduction velocity based on SEP was significantly improved in the ASC group compared to C and V groups (p< 0.05). Positive areas for Luxol fast blue staining were located at the injured site in the ASC group. Also, GNF-5 GFAP, Tuj-1 and NF160 were observed immunohistochemically in cells derived from implanted ASCs. These results suggested that improvement in neurological function by the transplantation of ASCs in dogs with spinal cord injury may be partially due to the neural differentiation of implanted stem cells. Keywords:adipose-derived stem cells, doggie, spinal cord injury, transplantation == Introduction == Central nervous system regeneration is usually highly limited after injury. Spinal cord injury (SCI) leads to cell death, particularly in GNF-5 neurons, oligodendrocytes, astrocytes, and precursor cells [12]. Any cavities and cysts resulting from this cell death and loss may interrupt axonal tracts. SCI culminates in glial scarring, a multifactorial process involving reactive astrocytes, glial progenitors, microglia, macrophages, fibroblasts, and Schwann cells [15]. Such scars are often oriented perpendicular to the neuraxis, contain transmembrane molecular inhibitors of axon growth, and appear impenetrable [34]. Neuronal phenotypes are not generated following SCI [42] and apparent lack of regenerative capacities of the adult spinal cords could result from the neurogenesis inhibitors of myelin-derived proteins, glial scar and extracellular matrix-derived factor [35]. Cell transplantation therapy using adult stem cells has recently been identified as a potential treatment for SCI [2]. Such cells can differentiate into appropriate neuronal phenotypes in ischemic or damaged brain and spinal cord [18]. Adipose tissue compartments are a particularly useful source of mesenchymal stem cells (MSCs) due to ease of harvest, clonogenic potential, and robust proliferative capacity [7]. Adipose-derived stem cells (ASCs) can differentiate into adipocyte, chondrocyte, myocyte, osteoblast, and even PLCB4 neural lineages [11]. ASCs may have therapeutic potential for neurological disorders, and functional recovery after transplantation of ASCs into the areas of spinal cord injuryin vivowas reported in rodent models of SCI [18]. Rodent spinal cords are smaller than canine cords and are also anatomically distinct in areas such as the extrapyramidal tract, therefore the rodent model is not suitable for detailed physical analyses or accurate evaluation of recovery [10]. Although it was reported that umbilical cord blood derived MSCs was effective in canine SCI model, there was little histological evidence of spinal cord tissue regeneration [22]. In this study, we examined whether canine ASCs could survive and integrate into neural cells and the effectiveness of canine ASCs around the improvement of GNF-5 neurological function in canine SCI model. == Materials and Methods == == Animals == Eleven healthy adult mixed-breed dogs (4.6 0.4 kg) were used. Applicable institutional and governmental regulations concerning the ethical use of animals were followed during the course of this research. This investigation was performed in accordance with the guidelines of the “Guide for the Care and Use of Laboratory Animals” of Seoul National University. SCI was induced by epidural ballon compression. The dogs were randomly assigned to 3 groups based on post-SCI treatment (31): Group C, control group with no ASCs transplantation (n = 3); Group V, vehicle group with phosphate-buffered saline (PBS) injection (n = 3); Group ASC, group with transplantation of allogenic ASCs into the site of SCI (n = 5). == Isolation and culture of ASCs == Adipose tissue was aseptically collected from the subcutaneous fat of a 2-year-old experimental doggie under anesthesia. Tissues were washed extensively with PBS, minced and digested with collagenase type I (1 mg/mL; Sigma, USA) at 37 for 2 h [16]. After washing with PBS and centrifuging at 4, pellets of stromal vascular fraction (SVF) were resuspended, filtered through 100 m nylon mesh and incubated overnight in DMEM with 10% fetal bovine serum (FBS; Gibco BRL, USA) at 37 with 5% humidified CO2. Unattached cells and residual non-adherent red blood cells were removed after 24 h by washing with PBS, and cell medium was exchanged with Keratinocyte-SFM (Gibco BRL, USA). The medium was supplemented with human recombinant epidermal growth factor (rEGF, 5 ng/mL; Gibco BRL, USA), bovine pituitary extract (50 g/mL; Gibco BRL, USA), 2 mM N-acetyl-L-cysteine (NAC; Sigma, USA), 0.2 mM L- ascorbic.