Clinical assessment after human adipose stem cell transplantation into dogs

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.

Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.

Studies on the anti-parasitic efficacy and safety of ivermectin and pyrantel pamoate compound against Dirofilaria immitis in dogs / 대한수의학회지

Dirofilaria (D.) immitis is an important canine parasitic nematode in dogs. D. immitis parasitizes the right ventricle and pulmonary artery of dogs. An ivermectin and pyrantel pamoate compound (IPPC) was administered to dogs naturally infected with this parasite. IPPC is composed of 68.0, 136.0 and 272.0 microg of ivermectin and 57.0, 114.0 and 227.0 mg pyrantel pamoate for small, middle, and large animals. Ivermectin has activity against nematodes and ectoparasites in dogs. Pyrantel pamoate is also effective against nematodes in dogs. Our results showed that this drug combination has good efficacy in D. immitis infected dogs.

Immunomodulatory effects of human amniotic membrane-derived mesenchymal stem cells

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.

The anti-parasitic efficacy of ivermectin and pyrantel pamoate compound against canine Toxocara canis and Trichuris vulpis / 대한수의학회지

Toxocara (T.) canis and Trichuris (T.) vulpis are very important canine parasitic nematodes. T. canis parasitize in small intestine and T. vulpis parasitize in large intestine. In order to control of these nematodes, ivermectin and pyrantel pamoate compound was applied to the dogs infected with these parasites naturally and artificially. This drug was composed of 68.0 microg of ivermectin and 57.0 mg of pyrantel pamoate for small animal, 136.0 microg of ivermectin and 114.0 mg of pyrantel pamoate for middle animal, and 272.0 microg of ivermectin and 227.0 mg of pyrantel pamoate for large animal. Ivermectin in this drug is activity to nematodes and ectoparisites. Pyrantel pamoate in this drug is also activity to nematodes. In this experiment, this drug had a good efficacy against T. canis and T. vulpis in the infected dogs.

Comparison of Human Muscle-Derived Stem Cells and Human Adipose-Derived Stem Cells in Neurogenic Trans-Differentiation

PURPOSE: Erectile dysfunction (ED) remains a major complication from cavernous nerve injury during radical prostatectomy. Recently, stem cell treatment for ED has been widely reported. This study was conducted to investigate the availability, differentiation into functional cells, and potential of human muscle-derived stem cells (hMDSCs) and human adipose-derived stem cells (hADSCs) for ED treatment. MATERIALS AND METHODS: We compared the neural differentiation of hMDSCs and hADSCs. Human muscle and adipose tissues were digested with collagenase, followed by filtering and centrifugation. For neural induction, isolated hMDSCs and hADSCs were incubated in neurobasal media containing forskolin, laminin, basic-fibroblast growth factor, and epidermal growth factor for 5 days. Following neural induction, hMDSCs and hADSCs were differentiated into neural cells, including neurons and glia, in vitro. RESULTS: In neural differentiated hMDSCs (d-hMDSCs) and differentiated hADSCs (d-hADSCs), neural stem cell marker (nestin) showed a significant decrease by immunocytochemistry, and neuronal marker (beta-tubulin III) and glial marker (GFAP) showed a significant increase, compared with primary hMDSCs and hADSCs. Real-time chain reaction analysis and Western blotting demonstrated significantly elevated levels of mRNA and protein of beta-tubulin III and GFAP in d-hADSCs compared with d-hMDSCs. CONCLUSIONS: We demonstrated that hMDSCs and hADSCs can be induced to undergo phenotypic and molecular changes consistent with neurons. The neural differentiation capacity of hADSCs was better than that of hMDSCs.

Establishment of Efficacy and Safety Assessment of Human Adipose Tissue-Derived Mesenchymal Stem Cells (hATMSCs) in a Nude Rat Femoral Segmental Defect Model

Human adipose tissue-derived mesenchymal stem cell (hATMSC) have emerged as a potentially powerful tool for bone repair, but an appropriate evaluation system has not been established. The purpose of this study was to establish a preclinical assessment system to evaluate the efficacy and safety of cell therapies in a nude rat bone defect model. Segmental defects (5 mm) were created in the femoral diaphyses and transplanted with cell media (control), hydroxyapatite/tricalcium phosphate scaffolds (HA/TCP, Group I), hATMSCs (Group II), or three cell-loading density of hATMSC-loaded HA/TCP (Group III-V). Healing response was evaluated by serial radiography, micro-computed tomography and histology at 16 weeks. To address safety-concerns, we conducted a GLP-compliant toxicity study. Scanning electron microscopy studies showed that hATMSCs filled the pores/surfaces of scaffolds in a cell-loading density-dependent manner. We detected significant increases in bone formation in the hATMSC-loaded HA/TCP groups compared with other groups. The amount of new bone formation increased with increases in loaded cell number. In a toxicity study, no significant hATMSC-related changes were found in body weights, clinical signs, hematological/biochemical values, organ weights, or histopathological findings. In conclusion, hATMSCs loaded on HA/TCP enhance the repair of bone defects and was found to be safe under our preclinical efficacy/safety hybrid assessment system.

In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors

The homing properties of adipose tissue-derived mesenchymal stem cells (AdMSCs) have stimulated intravenous applications for their use in stem cell therapy. However, the soluble factors and corresponding cellular receptors responsible for inducing chemotaxis of AdMSCs have not yet been reported. In the present study, the migration capacity of human AdMSCs (hAdMSCs) toward various cytokines or growth factors (GFs) and the expression of their receptors were determined. In a conventional migration assay, PDGF-AB, TGF-beta1, and TNF-alpha showed the most effective chemoattractant activity. When AdMSCs were preincubated with various chemokines or GF, and then allowed to migrate toward medium containing 10% FBS, those preincubated with TNF-alpha showed the highest migratory activity. Next, hAdMSCs were either preincubated or not with TNF-alpha, and allowed to migrate in response to various GFs or chemokines. Prestimulation with TNF-alpha increased the migration activity of hAdMSCs compared to unstimulated hAdMSCs. When analyzed by FACS and RT-PCR methods, hAdMSCs were found to express C-C chemokine receptor type 1 (CCR1), CCR7, C-X-C chemokine receptor type 4 (CXCR4), CXCR5, CXCR6, EGF receptor, fibroblast growth factor receptor 1, TGF-beta receptor 2, TNF receptor superfamily member 1A, PDGF receptor A and PDGF receptor B at both the protein and the mRNA levels. These results indicate that the migration capacity of hAdMSCs is controlled by various GFs and chemokines. Prior in vitro modulation of the homing capacity of hAdMSCs could stimulate their movement into injured sites in vivo when administered intravenously, thereby improving their therapeutic potential.

In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors

The homing properties of adipose tissue-derived mesenchymal stem cells (AdMSCs) have stimulated intravenous applications for their use in stem cell therapy. However, the soluble factors and corresponding cellular receptors responsible for inducing chemotaxis of AdMSCs have not yet been reported. In the present study, the migration capacity of human AdMSCs (hAdMSCs) toward various cytokines or growth factors (GFs) and the expression of their receptors were determined. In a conventional migration assay, PDGF-AB, TGF-beta1, and TNF-alpha showed the most effective chemoattractant activity. When AdMSCs were preincubated with various chemokines or GF, and then allowed to migrate toward medium containing 10% FBS, those preincubated with TNF-alpha showed the highest migratory activity. Next, hAdMSCs were either preincubated or not with TNF-alpha, and allowed to migrate in response to various GFs or chemokines. Prestimulation with TNF-alpha increased the migration activity of hAdMSCs compared to unstimulated hAdMSCs. When analyzed by FACS and RT-PCR methods, hAdMSCs were found to express C-C chemokine receptor type 1 (CCR1), CCR7, C-X-C chemokine receptor type 4 (CXCR4), CXCR5, CXCR6, EGF receptor, fibroblast growth factor receptor 1, TGF-beta receptor 2, TNF receptor superfamily member 1A, PDGF receptor A and PDGF receptor B at both the protein and the mRNA levels. These results indicate that the migration capacity of hAdMSCs is controlled by various GFs and chemokines. Prior in vitro modulation of the homing capacity of hAdMSCs could stimulate their movement into injured sites in vivo when administered intravenously, thereby improving their therapeutic potential.