Yasuji Harada

Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells

BackgroundBone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.ResultsSamples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.ConclusionOur results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

Embryonic Stem Cells form Articular Cartilage, Not Teratomas, in Osteochondral Defects of Rat Joints

Embryonic stem (ES) cells are considered to be a potential tool for repairing articular cartilage defects, but so far it has been impossible to cause these cells to differentiate into chondrocytes exclusively, either in vivo or in vitro. To explore a potential new cell source of cell transplantation for articular cartilage defects, we transplanted ES cells into articular cartilage defects in immunosuppressed rats. ES cells (AB2.2 or CCE cells) were transplanted into articular cartilage defects in the patellar groove of immunosuppressed rats treated with cyclosporine. The cells were histologically observed until 8 weeks after transplantation. To determine whether the repair tissue in the defect in the AB2.2-transplanted group was derived from the transplanted cells, the neomycin-resistant gene, which had been transfected into AB2.2 cells but does not exist in rat cells, was used for detection. The cells produced cartilage, resulting in repair of the defects from 4 weeks until 8 weeks after the transplantation without forming any teratomas. The neomycin-resistant gene was detected in every sample, demonstrating that the repair tissue in the AB2.2-transplanted group was derived from the transplanted AB2.2 cells. The environment of osteochondral defects is chondrogenic for ES cells. ES cells may thus be a potential tool for repairing articular cartilage defects.

Safety of Autologous Bone Marrow Stromal Cell Transplantation in Dogs with Acute Spinal Cord Injury

OBJECTIVE To assess the feasibility and safety of transplantation of autologous bone marrow stromal cell (BMSC) in dogs with acute spinal cord injury (SCI). STUDY DESIGN An open-label single-arm trial. ANIMALS Dogs (n = 7) with severe SCI from T6 to L5, caused by vertebral fracture and luxation. METHODS Decompressive and stabilization surgery was performed on dogs with severe SCI caused by vertebral fracture and luxation. Autologous BMSCs were obtained from each dogs femur, cultured, and then injected into the lesion in the acute stage. Adverse events and motor and sensory function were observed for >1 year after SCI. RESULTS Follow-up was 29-62 months after SCI. No complications (eg, infection, neuropathic pain, worsening of neurologic function) were observed. Two dogs walked without support, but none of the 7 dogs had any change in sensory function. CONCLUSIONS Autologous BMSC transplantation is feasible and safe in dogs with acute SCI. Further studies are needed to determine the efficacy of this therapy.

Cytokine-immobilized microparticle-coated plates for culturing hematopoietic progenitor cells.

The purpose of this study was to provide a culture method for an effective expansion of human CD 34 positive hematopoietic progenitor cells (CD 34 (+) HCs) utilizing low molecular weight heparin/protamine microparticles (LH/P MPs) which can be stably coated onto plastic surfaces and cytokines. CD 34 (+) HCs optimally proliferated on LH/P MP-coated plates in the presence of stem cell factor (SCF; 5 ng/ml), thrombopoietin (Tpo; 10 ng/ml), and Flt-3 ligand (Flt-3; 10 ng/ml) in hematopoietic progenitor growth medium (HPGM). After 6 days, the total cells expanded 16.5-fold. Those cytokines were shown to be partially immobilized on the LH/P MP-coated plates, and the immobilized cytokines were gradually released into the medium with half releasing time of 3-4 days. Since flow cytometry analyses revealed that 90% of initial cells and 44.5% of expanded cells were CD 34 positive, CD 34 (+) HCs were estimated to have increased 8.0-fold after 6 days, and to have increased to over 31.9-fold after 12 days. In contrast, cultured CD 34 (+) HCs on non-coated tissue culture plates increased only 2.9-fold in the identical medium after 6 days, and only 5.2-fold after 12 days.

Fragmin/Protamine Microparticle‐Coated Matrix Immobilized Cytokines to Stimulate Various Cell Proliferations With Low Serum Media

Fragmin/protamine microparticles (F/P MPs) have been shown to bind to culture plates, thereby retaining heparin-binding cytokines. Most protocols for in vitro cultures of human microvascular endothelial cells (hMVECs), human dermal fibroblast cells (hDFCs), and hematopoietic cell line (TF-1) include high fetal bovine serum (FBS) (10%) medium as a nutritional supplement. Growth rates of those cells on the F/P MP-coated plates were higher in low FBS (1%) medium containing fibroblast growth factor (FGF)-2 (for hMVECs and hDFCs) and interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (for TF-1 cells) than without coating. The cytokines in low FBS medium were shown to be immobilized on the F/P MP-coated plate and released into the culture medium with a half releasing time of 4-5 days. Furthermore, those cells grew well on each cytokine-preimmobilized F/P MP-coated plate in low FBS medium. Thus, the F/P MP-coated matrix with adequate heparin-binding cytokines may provide biomaterials for controlling cellular growth and differentiation.

Effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs.

OBJECTIVE To evaluate effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs. ANIMALS 12 healthy female Beagles. PROCEDURES A mid-diaphyseal transverse osteotomy (stabilized with an intramedullary pin) of the right tibia was performed in each dog. The carprofen group (n = 6 dogs) received carprofen (2.2 mg/kg, PO, q 12 h) for 120 days; the control group (6) received no treatment. Bone healing and change in callus area were assessed radiographically over time. Dogs were euthanized 120 days after surgery, and tibiae were evaluated biomechanically and histologically. RESULTS The osteotomy line was not evident in the control group on radiographs obtained 120 days after surgery. In contrast, the osteotomy line was still evident in the carprofen group. Callus area was significantly less in the carprofen group, compared with the area in the control group, at 20, 30, and 60 days after surgery. At 120 days after surgery, stiffness, elastic modulus, and flexural rigidity in the carprofen group were significantly lower than corresponding values in the control group. Furthermore, histologic evaluation revealed that the cartilage area within the callus in the carprofen group was significantly greater than that in the control group. CONCLUSIONS AND CLINICAL RELEVANCE Long-term administration of carprofen appeared to inhibit bone healing in dogs that underwent tibial osteotomy. We recommend caution for carprofen administration when treating fractures that have delays in healing associated with a reduction in osteogenesis as well as fractures associated with diseases that predispose animals to delays of osseous repair.

Use of Controlled Mechanical Stimulation in Vivo to Induce Cartilage Layer Formation on the Surface of Osteotomized Bone

A micromachine was used to study the response of mesenchymal tissue to mechanically controlled motion in vivo. The middle portion of the coccygeal vertebra of Fischer 344 rats was osteotomized, and continuous bending motion was applied for 4 weeks. The experimental groups were divided into two groups with higher sliding displacement applied at the osteotomized gap of group II. Hyaline cartilage tissue was generated at the osteotomized ends, and was predominantly formed on the side that extended during the bending motion. These newly formed tissues stained intensively with safranin O and toluidine blue, positively with immunostain for type II collagen, but negatively with immunostain for type I collagen. Articular cartilage-like tissues with a surface and a layer structure were obtained in group II, in which higher sliding motion was applied. Light and electron microscopy revealed morphological features similar to those of normal articular cartilage tissue in the superficial and middle zones of the tissues obtained in group II. Collagen fibrils in the superficial zone were found aligned parallel to the smooth surface. Although tidemark formation was not observed in the deep zone, the structure was much more natural than that of any other tissue-engineered cartilage reported to date. These results suggest that controlled sliding stimulation can elicit the generation of articular cartilage structure in vivo.

Human Stem Cell Factor (SCF) is a Heparin-Binding Cytokine

Binding affinities of chemically modified heparins for human stem cell factor (SCF) were examined using fragmin/protamine microparticles (F/P MPs) and an enzyme-linked immunosorbent assay (ELISA). The binding of SCF to F/P MP-coated plates was inhibited with high concentrations of heparin and fragmin, but not others. The binding of SCF was also inhibited with 0.55 M or higher concentrations of NaCl in the medium. These results suggested that a high content of all three sulfate groups in repeating disaccharide units is required for interaction with SCF. Furthermore, pre-immobilized SCF on F/P MP-coated plates significantly stimulated proliferation of a human erythroleukemia cell line.

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic-pituitary axis.

Cushings disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelsons syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary-adrenal axis in clinically normal dogs. Dogs were administered 5mg/kg trilostane twice a day every day for 8 weeks (n=8) or 16 weeks (n=3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushings disease.

Effects of small intestinal ischemia and reperfusion on expression of tumor necrosis factor-α and interleukin-6 messenger RNAs in the jejunum, liver, and lungs of dogs

OBJECTIVE To determine the effects of intestinal ischemia and reperfusion on the expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNAs in the jejunum, liver, and lungs of dogs. ANIMALS 8 healthy adult Beagles. PROCEDURES In each dog, the cranial mesenteric artery was occluded for 0 (control group; n=4) or 60 (I-R group; 4) minutes, followed by reperfusion for 480 minutes; serum TNF-alpha and IL-6 activities and expression levels of TNF-alpha and IL-6 mRNAs in jejunal, hepatic, and lung tissues were measured before and at the end of the ischemic period and at intervals during reperfusion. For each variable, values were compared between the control and I-R groups at each time point. RESULTS Compared with the control group, serum IL-6 activity increased significantly after 180 minutes of reperfusion in the I-R group; also, jejunal TNF-alpha mRNA expression increased significantly after 60 (peak) and 180 minutes of reperfusion. In the I-R group, expressions of IL-6 mRNA in the liver and TNF-alpha and IL-6 mRNAs in the lungs increased significantly at 480 minutes of reperfusion, compared with the control group. Serum TNF-alpha activity, expression of IL-6 mRNA in the jejunum, and expression of TNF-alpha mRNA in the liver in the control and I-R groups did not differ. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the liver, lungs, and jejunum contributed to the production of TNF-alpha and IL-6 after intestinal ischemia and reperfusion in dogs, suggesting that intestinal ischemia and reperfusion induce a systemic proinflammatory cytokine response in dogs.