In this study, we evaluated if the implantation of allogenic adipose-derived stem cells (ASCs) improved neurological function in a canine spinal cord injury model. Eleven adult dogs were assigned to three groups according to treatment after spinal cord injury by epidural balloon compression: C group (no ASCs treatment as control), V group (vehicle treatment with PBS), and ASC group (ASCs treatment). ASCs or vehicle were injected directly into the injured site 1 week after spinal cord injury. Pelvic limb function after transplantation was evaluated by Olby score. Magnetic resonance imaging, somatosensory evoked potential (SEP), histopathologic and immunohistichemical examinations 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, 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.

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with β-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.

This study was to determine the effects of allogenic umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) and recombinant methionyl human granulocyte colony-stimulating factor (rmhGCSF) on a canine spinal cord injury model after balloon compression at the first lumbar vertebra. Twenty-five adult mongrel dogs were assigned to five groups according to treatment after a spinal cord injury: no treatment (CN); saline treatment (CP); rmhGCSF treatment (G); UCB-MSCs treatment (UCB-MSC); co-treatment (UCBG). The UCB-MSCs isolated from cord blood of canine fetuses were prepared as 106 cells/150 µl saline. The UCB-MSCs were directly injected into the injured site of the spinal cord and rmhGCSF was administered subcutaneously 1 week after the induction of spinal cord injury. The Olby score, magnetic resonance imaging, somatosensory evoked potentials and histopathological examinations were used to evaluate the functional recovery after transplantation. The Olby scores of all groups were zero at the 0-week evaluation. At 2 week after the transplantation, the Olby scores in the groups with the UCB-MSC and UCBG were significantly higher than in the CN and CP groups. However, there were no significant differences between the UCB-MSC and UCBG groups, and between the CN and CP groups. These comparisons remained stable at 4 and 8 week after transplantation. There was significant improvement in the nerve conduction velocity based on the somatosensory evoked potentials. In addition, a distinct structural consistency of the nerve cell bodies was noted in the lesion of the spinal cord of the UCB-MSC and UCBG groups. These results suggest that transplantation of the UCB-MSCs resulted in recovery of nerve function in dogs with a spinal cord injury and may be considered as a therapeutic modality for spinal cord injury.

Liver transplantation is the last resort for liver failure patients. However, due to the shortage of donor organs, bioengineered liver generated from decellularized whole liver scaffolds and induced pluripotent stem cell (iPSC)-derived hepatocytes (iPSC-Heps) is being studied as an alternative approach to treat liver disease. Nevertheless, there has been no report on both the interaction of iPSC-Heps with a liver extracellular matrix (ECM) and the analysis of recellularized iPSC-Heps into the whole liver scaffolds. In this study, we produced porcine iPSC-Heps, which strongly expressed the hepatic markers α-fetoprotein and albumin and exhibited hepatic functionalities, including glycogen storage, lipid accumulation, low-density lipoprotein uptake, and indocyanine green metabolism. Supplementation of ECM from porcine decellularized liver containing liver-derived growth factors stimulated the albumin expression of porcine iPSC-Heps during differentiation procedures. The iPSC-Heps were reseeded into decellularized liver scaffolds, and the recellularized liver was cultured using a continuous perfusion system. The recellularized liver scaffolds were transplanted into rats for a short term, and the grafts expressed hepatocyte markers and did not rupture. These results provide a foundation for development of bioengineered liver using stem cell and decellularized scaffolds.

ABSTRACT. Previous animal studies have shown that transplantation of mesenchymal stem cells (MSCs) into spinal cord lesions enhances axonal regeneration and promotes functional recovery. We isolated the MSCs derived from fat, bone marrow, Wharton's jelly and umbilical cord blood (UCB) positive for MSC markers and negative for hematopoietic cell markers. Their effects on the regeneration of injured canine spinal cords were compared. Spinal cord injury was induced by balloon catheter compression. Dogs with injured spinal cords were treated with only matrigel or matrigel mixed with each type of MSCs. Olby and modified Tarlov scores, immunohistochemistry, ELISA and Western blot analysis were used to evaluate the therapeutic effects. The different MSC groups showed significant improvements in locomotion at 8 weeks after transplantation (P<0.05). This recovery was accompanied by increased numbers of surviving neuron and neurofilament-positive fibers in the lesion site. Compared to the control, the lesion sizes were smaller, and fewer microglia and reactive astrocytes were found in the spinal cord epicenter of all MSC groups. Although there were no significant differences in functional recovery among the MSCs groups, UCB-derived MSCs (UCSCs) induced more nerve regeneration and anti-inflammation activity (P<0.05). Transplanted MSCs survived for 8 weeks and reduced IL-6 and COX-2 levels, which may have promoted neuronal regeneration in the spinal cord. Our data suggest that transplantation of MSCs promotes functional recovery after SCI. Furthermore, application of UCSCs led to more nerve regeneration, neuroprotection and less inflammation compared to other MSCs.

IntroductionMesenchymal stem cells can potentially be used in therapy for spinal cord injury (SCI). Methylprednisolone sodium succinate (MPSS) has been used as a scavenging agent in acute SCI treatment, but its use no longer recommended. This study aimed to identify ways to reduce the usage and risk of high doses of glucocorticoid steroids, and determine whether AD-MSCs could be used as an early alternative treatment modality for acute SCI.MethodsSixteen adult beagle dogs with SCI were assigned to four treatment groups: control, MPSS, AD-MSCs, and AD-MSCs + MPSS. Additionally, one dog was used to evaluate the distribution of AD-MSCs in the body after injection. AD-MSCs (1 × 107 cells) were injected intravenously once a day for 3 days beginning at 6 hours post-SCI. MPSS was also injected intravenously according to the standard protocol for acute SCI. A revised Tarlov scale was used to evaluate hindlimb functional recovery. The levels of markers for oxidative metabolism (3-nitrotyrosine, 4-hydroxynonenal, and protein carbonyl) and inflammation (cyclooxygenase-2, interleukin-6, and tumor necrosis factor-α) were also measured.ResultsAt 7 days post-treatment, hindlimb movement had improved in the AD-MSCs and AD-MSCs + MPSS groups; however, subjects in the groups treated with MPSS exhibited gastrointestinal hemorrhages. Hematoxylin and eosin staining revealed fewer hemorrhages and lesser microglial infiltration in the AD-MSCs group. The green fluorescent protein-expressing AD-MSCs were clearly detected in the lung, spleen, and injured spinal cord; however, these cells were not detected in the liver and un-injured spinal cord. Levels of 3-nitrotyrosine were decreased in the MPSS and AD-MSCs + MPSS groups; 4-hydroxynenonal and cyclooxygenase-2 levels were decreased in all treatment groups; and interleukin-6, tumor necrosis factor-α, and phosphorylated-signal transducer and activator transcription 3 levels were decreased in the AD-MSCs and AD-MSCs + MPSS groups.ConclusionOur results suggest that early intravenous injection of AD-MSCs after acute SCI may prevent further damage through enhancement of antioxidative and anti-inflammatory mechanisms, without inducing adverse effects. Additionally, this treatment could also be used as an alternative intravenous treatment modality for acute SCI.

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Canine mesenchymal stem cells (cMSCs) derived from umbilical cord blood represent a potentially useful source of stem cells for therapy. The aim of this study was to compare the effects of different transplantation times of cMSCs after spinal cord injury (SCI). A total of 21 dogs were subjected to SCI by balloon-induced compression of the first lumbar vertebrae for 12 h. Of the 21 dogs, 12 were divided into four groups of three according to the time of stem cell (1 × 10 6 ) transplantation at the injury site: control no treatment, 12 h, 1 week, and 2 weeks. The remaining 9 animals were negative harvest (HA) time controls for each treatment group (n = 3). Olby and Tarlov scores were used to evaluate functional recovery of the hindlimbs. Markers for neuronal regeneration (Tuj-1, nestin, MAP2, and NF-M), astrogliosis (GALC, GFAP, and pSTAT3), signal molecules for actin cytoskeleton (RhoA, Cdc42, and Rac1), inflammation (COX-2), and neurotrophins (NT-3) were evaluated by Western blot analysis. Scores of the 1-week transplantation group showed significant improvement compared to controls. Hematoxylin and eosin (H&E) staining revealed less fibrosis at the injury site in the 1-week transplantation group compared to other groups and immunohistochemistry showed increased expression of neuronal markers. Furthermore, in both 1-week and 2-week transplantation groups, Tuj-1, nestin, MAP2, NF-M, NT-3, and GFAP increased, but pSTAT3, GALC, and COX2 decreased. RhoA decreased and Rac1 and Cdc42 increased in the 1-week transplantation group. In conclusion, transplantation of cMSCs 1 week after SCI was more effective in improving clinical signs and neuronal regeneration and reducing fibrosis formation compared to the other transplantation times evaluated. Subsequently, these data may contribute to the optimization of timing for MSC transplantation used as a therapeutic modality.Key words: Dog; Stem cells; Optimal transplantation time; Spinal cord injury INTRODUCTIONcomposed of connective tissue elements and glial cells such as astrocytes and oligodendrocytes were thought to explain the failure of central nervous system (CNS) Responses to spinal cord injury (SCI) include disruption of the spinal cord vasculature, ischemia, glutamaterregeneration after injury (6). Oligodendrocytes provide myelin sheaths for enhanced axonal transmission (6), gic excitotoxicity, oxidative cell stress, lipid peroxidation, inflammation, and scar formation (30,51). Inflammation and astrocytes are important for neurotransmitter regulation and ion homeostasis (6,47). But after injury they or scarring as a result of SCI has been found to be detrimental to nerve regeneration (5,6,42). At the site of inare the major cell type responsible for walling off areas of damage to protect normal tissue from further erosion. flammation in the spinal cord, cyclooxygenase-2 (COX2) expression is markedly increased (21,40) and glial cellsIn addition, astrocytes respond to multiple extracellular signaling molecules through a complex assortment of ininteract with inflammator...