Jin-Ok Ahn

Anti-tumor effects of canine adipose tissue-derived mesenchymal stromal cell-based interferon-β gene therapy and cisplatin in a mouse melanoma model

BACKGROUND AIMS Adipose tissue (AT)-derived mesenchymal stromal cells (MSC) (AT-MSC) represent a novel tool for delivering therapeutic genes to tumor cells. Interferon (IFN)-β is a cytokine with pleiotropic cellular functions, including anti-proliferative, immunomodulatory and anti-angiogenic activities. The purpose of this study was to engineer canine AT-MSC (cAT-MSC) producing IFN-β and to evaluate the anti-tumor effect of cAT-MSC-IFN-β combined with cisplatin in mouse melanoma model. METHODS cAT-MSC engineered to express mouse IFN-β were generated using a lentiviral vector (cAT-MSC-IFN-β) and the secreted IFN-β-induced inhibition of tumor cell growth and apoptosis on B16F10 cells was investigated in vitro prior to in vivo studies. Melanoma-bearing mouse was developed by injecting B16F10 cells subcutaneously into 6-week-old C57BL/6 mice. After 14 days, cisplatin (10 mg/kg) was injected intratumorally, and 3 days later the engineered cAT-MSC were injected subcutaneously every 3 days to death. Tumor volume and survival times were measured. RESULTS The combination treatment of cAT-MSC-IFN-β with cisplatin was more effective in inhibiting the growth of melanoma and resulted in significantly extended survival time than both an unengineered cAT-MSC-cisplatin combination group and a cisplatin-alone group. Interestingly, subcutaneously injected cAT-MSC-IFN-β were migrated to tumor sites. CONCLUSIONS Our data suggest that canine AT-MSC could serve as a powerful cell-based delivery vehicle for releasing therapeutic proteins to tumor lesions. Maximal anti-tumor effects were seen when this therapy was combined with a DNA-damaging chemotherapeutic agent. This study demonstrates the possible applicability of AT-MSC-mediated IFN-β in treating canine and human cancer patients.

A sub-Tenon's capsule injection of lidocaine induces extraocular muscle akinesia and mydriasis in dogs.

The effect of local anaesthetic on the extraocular muscles was investigated in dogs by injecting lidocaine into the space between Tenons capsule and the sclera. A cross-over design was used with both eyes from five Beagle dogs randomly injected, under general anaesthesia, with 1 mL of 2% lidocaine (1 mL-lidocaine group), 2 mL of 2% lidocaine (2 mL-lidocaine group) or 2 mL of normal saline (control group). Each eye was assigned to all treatments with a minimum 14 day interval between injections. Changes in eye position, pupil diameter, and intraocular pressure (IOP) were evaluated during the procedure. All eyes in the 2 mL-lidocaine group exhibited akinesia and mydriasis (pupil diameter >10mm) with an onset time of 6.5 ± 4.9 and 4.2 ± 4.3 min, respectively. In the 1 mL-lidocaine group, akinesia was induced in nine eyes and mydriasis occurred in seven eyes at 10.7 ± 5.8 and 5.4 ± 2.4 min after the injection, respectively. No changes in eye position or pupil diameter were observed in the control group. Akinesia was maintained for 44.3 ± 26.7 min in the 1 mL-lidocaine group and for 88.5 ± 17.2 min in the 2 mL-lidocaine group. Duration of mydriasis was 51.7 ± 28.9 min in the 1 mL-lidocaine group and 82.9 ± 15.6 min in the 2 mL-lidocaine group. Marked chemosis and sub-conjunctival haemorrhage occurred in 16/30 and 15/30 eyes, respectively. No significant change in IOP was observed between the mean pre- and post-injection values in all groups. These results suggest that a sub-Tenons injection of 2 mL of 2% lidocaine provided effective extraocular muscle akinesia and mydriasis in dogs.

TSG-6 Secreted by Human Adipose Tissue-derived Mesenchymal Stem Cells Ameliorates DSS-induced colitis by Inducing M2 Macrophage Polarization in Mice

Previous studies have revealed that mesenchymal stem cells (MSCs) alleviate inflammatory bowel disease (IBD) by modulating inflammatory cytokines in the inflamed intestine. However, the mechanisms underlying these effects are not completely understood. We sought to investigate the therapeutic effects of human adipose tissue-derived (hAT)-MSCs in an IBD mouse model and to explore the mechanisms of the regulation of inflammation. Dextran sulfate sodium-induced colitis mice were infused with hAT-MSCs intraperitoneally and colon tissues were collected on day 10. hAT-MSCs were shown to induce the expression of M2 macrophage markers and to regulate the expression of pro- and anti-inflammatory cytokines in the colon. Quantitative real time-PCR analyses demonstrated that less than 20 hAT-MSCs, 0.001% of all intraperitoneally injected hAT-MSCs, were detected in the inflamed colon. To investigate the effects of hAT-MSC-secreted factors in vitro, transwell co-culture system was used, demonstrating that tumour necrosis factor-α-induced gene/protein 6 (TSG-6) released by hAT-MSCs induces M2 macrophages. In vivo, hAT-MSCs transfected with TSG-6 small interfering RNA, administered intraperitoneally, were not able to induce M2 macrophage phenotype switch in the inflamed colon and had no significant effects on IBD severity. In conclusion, hAT-MSC-produced TSG-6 can ameliorate IBD by inducing M2 macrophage switch in mice.

C-reactive protein as an indicator of inflammatory responses to experimentally induced cystitis in dogs

The aim of this study was to demonstrate and assess C-reactive protein (CRP) changes in dogs with induced bacterial cystitis with or without antibiotics. We also evaluated availability of CRP levels to serve as an indicator for monitoring or diagnosing bacterial cystitis. Serial CRP concentrations in dogs with induced bacterial cystitis were higher than those of controls (p < 0.001). CRP concentrations peaked on day 7 and gradually decreased thereafter. In the treatment group, CRP concentrations decreased after medication compared to the untreated group (p = 0.032). CRP levels had a linear correlation with urine white blood cell counts among all groups (r = 0.837, p < 0.001, n = 140). Compared to the negative urine culture group, dogs with positive urine culture results had higher CRP concentrations (median 43.8 mg/L vs. 5.9 mg/L; p < 0.001). Area under the receiver operating characteristic curve was 0.955; when cut-off value was 12.2 mg/L, CRP measurements were found to have a sensitivity of 92.3% and specificity of 86.4%. This result indicates that rapid increases of CRP occurred after inducing bacterial cystitis and CRP may be a useful indicator for monitoring or diagnosing canine bacterial cystitis together with sediment urinalysis and urine bacterial culture.

Enhanced Hepatogenic Transdifferentiation of Human Adipose Tissue Mesenchymal Stem Cells by Gene Engineering with Oct4 and Sox2

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase “transdifferentiation” of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation.

Effect of ectopic OCT4 expression on canine adipose tissue-derived mesenchymal stem cell proliferation.

Enhancing the proliferative capacity of mesenchymal stem cells (MSCs) is critical for increasing their therapeutic potential in a variety of diseases. We hypothesized that lentivirus‐mediated overexpression of canine octamer‐binding transcription factor 4 (OCT4) might influence the proliferation of canine adipose tissue‐derived MSCs (cATMSCs). cOCT4‐cATMSCs were generated by transducing cATMSCs with a cOCT4‐lentiviral vector. Increased expression of cOCT4 was confirmed using RT‐PCR and immunoblotting. Immunophenotypic characterization using flow cytometry indicated that the CD29, CD44, CD73, CD90, and CD105 surface markers were highly expressed by both cOCT4‐ and mock‐transduced cATMSCs (mock‐cATMSCs), whereas the CD31 and CD45 markers were absent. We performed the osteogenic differentiation assay to evaluate the effects of cOCT4 overexpression on the osteogenic differentiation potential of cATMSCs. The results showed that cOCT4‐cATMSCs had a much higher potential for osteogenic differentiation than mock‐cATMSCs. Next, the proliferative capacities of cOCT4‐ and mock‐cATMSCs were evaluated using a WST‐1 cell proliferation assay and trypan blue exclusion. cOCT4‐cATMSCs showed a higher proliferative capacity than mock‐cATMSCs. Cell cycle analysis indicated that overexpression of cOCT4 in cATMSCs induced an increase in the proportion of cells in S and G2/M phases. Consistent with this, immunoblot analysis showed that cyclin D1 expression was increased in cOCT4‐cATMSCs. In conclusion, our results indicate that lentivirus‐mediated overexpression of cOCT4 increased the proliferative capacity of cATMSCs. OCT4‐mediated enhancement of cell proliferation may be a useful method for expanding MSC population rapidly without loss of stemness.

Immunomodulatory effects of soluble factors secreted by feline adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) have immunomodulatory functions and differentiation capacity, and their clinical use is increasing in veterinary species. Although MSCs have been applied in the treatment in various inflammatory diseases, mechanistic research on feline MSCs is lacking. Accordingly, in this study, we aimed to investigate the immunomodulatory mechanisms of MSCs isolated from feline adipose tissue (fATMSCs). fATMSCs from healthy cats were cultured in an appropriate manner and cocultured with transwell-separated allogeneic feline peripheral blood mononuclear cells (fPBMCs) and RAW264.7 murine macrophages. After 48h of coculture, RNA was extracted from RAW264.7 cells and fPBMCs. Cytokine expression in these cells was measured using quantitative real-time polymerase chain reaction (qRT-PCR) and compared according to the presence of fATMSCs. The mRNA levels of pro-inflammatory cytokines, e.g., tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase, and interleukin (IL)-1β, were significantly decreased in cocultures of mitogen-stimulated RAW264.7 cells with fATMSCs compared with that in the RAW264.7 cells control group. Additionally, changes in the expression of mRNAs extracted from fPBMCs were as follows: pro-inflammatory TNF-α, interferon-γ, and IL-6 were decreased, and anti-inflammatory IL-10 was increased during coculture of mitogen-stimulated allogeneic fPBMCs with fATMSCs. We also extracted RNA and collected supernatants from fATMSCs during transwell culture for measurement of the expression and secretion of soluble factors by qRT-PCR and enzyme-linked immunosorbent assays, respectively. The mRNA expression of immunomodulatory factors from fATMSCs, including cyclooxygenase-2 (COX-2), transforming growth factor (TGF)-β, indoleamine-2,3-dioxygenase (IDO) and hepatocyte growth factor, increased in the presence of RAW264.7 cells. Similarly, TGF-β, COX-2, and IDO mRNA expression and prostaglandin E2 (PGE2) secretion from fATMSCs increased in the presence of allogeneic fPBMCs. Finally, we measured the viability of fPBMCs under various conditions. Cell viability decreased in fPBMCs suspended in fATMSC-derived conditioned medium, and this reduction was alleviated in the group supplemented with NS-398 a PGE2 inhibitor. Our data suggested that soluble factors, including PGE2, secreted by fATMSCs played an important role in the immunomodulatory effects of these cells. These findings may be helpful in the application of fATMSCs to feline patients with immune-related diseases.

Differences of RNA Expression in the Tendon According to Anatomic Outcomes in Rotator Cuff Repair

Background: Despite increased understanding of the pathophysiology of rotator cuff tears and the evolution of rotator cuff repair, healing failure remains a substantial problem. The critical roles played by biological factors have been emphasized, but little is known of the implications of gene expression profile differences at the time of repair. Purpose: To document the relationship between the perioperative gene expression of healed and unhealed rotator cuffs by RNA microarray analysis. Study Design: Case-control study; Level of evidence, 3. Methods: Superior (supraspinatus involvement) and posterosuperior (supraspinatus and infraspinatus involvement) tears were included in the study. Samples of rotator cuff tendons were prospectively collected during rotator cuff surgery. Three samples were harvested at the tendon ends of tears from the anterior, middle (apex), and posterior parts using an arthroscopic punch. Seven patients with an unhealed rotator cuff were matched one-to-one with patients with a healed rotator cuff by sex, age, tear size, and fatty degeneration of rotator cuff muscles. mRNA microarray analysis was used to identify genetic differences between healed and unhealed rotator cuff tendons. Gene ontology and gene association files were obtained from the Gene Ontology Consortium, and the Gene Ontology system in DAVID was used to identify enhanced biological processes. Results: Microarray analyses identified 262 genes that were differentially expressed by at least 1.5-fold between the healed and unhealed groups. Overall, in the healed group, 103 genes were significantly downregulated, and 159 were significantly upregulated. DAVID Functional Annotation Cluster analysis showed that in the healed group, the genes most upregulated were related to the G protein–coupled receptor protein signaling pathway and to the neurological system. On the other hand, the genes most downregulated were related to immune and inflammatory responses. BMP5 was the gene most upregulated in the healed group, and the majority of downregulated genes were involved in the immune/inflammatory response. Conclusion: The downregulation of inflammatory response genes and the upregulation of cell differentiation genes in torn rotator cuffs at the time of surgery are related to rotator cuff healing. These results provide useful baseline information for future biological studies on rotator cuff healing.

TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice

BackgroundInflammatory bowel disease (IBD) is an intractable autoimmune disorder that markedly deteriorates one’s quality of life. Mesenchymal stem cells (MSCs) alleviate inflammation by modulating inflammatory cytokines in inflamed tissues, and have been suggested as a promising alternative for IBD treatment in human and veterinary cases. Furthermore, tumor necrosis factor-α-induced gene/protein 6 (TSG-6) is a key factor influencing MSC immunomodulatory properties; however, the precise mechanism of TSG-6 release from canine MSCs in IBD remains unclear. This study aimed to assess the therapeutic effects of canine adipose tissue-derived (cAT)-MSC-produced TSG-6 in an IBD mouse model and to explore the mechanisms underlying the immunomodulatory properties.MethodsMice with dextran sulfate sodium-induced colitis were administered cAT-MSCs intraperitoneally; colon tissues were collected on day 10 for histopathological, quantitative real-time polymerase chain reaction, and immunofluorescence analyses.ResultscAT-MSC-secreted TSG-6 ameliorated IBD and regulated colonic expression of pro- and anti-inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-10. To investigate the effect of cAT-MSC-secreted TSG-6 on activated macrophages in vitro, a transwell coculture system was used; TSG-6 released by cAT-MSCs induced a macrophage phenotypic switch from M1 to M2. The cAT-MSC-secreted TSG-6 increased M2 macrophages in the inflamed colon in vivo.ConclusionsTSG-6 released from cAT-MSCs can alleviate dextran sulfate sodium-induced colitis by inducing a macrophage phenotypic switch to M2 in mice.

Canine mesenchymal stem cells treated with TNF-α and IFN-γ enhance anti-inflammatory effects through the COX-2/PGE2 pathway

Mesenchymal stem cells (MSCs) have been used in studies on treatment of various diseases, and their application to immune-mediated diseases has garnered interest. Various methods for enhancing the immunomodulation effect of human MSCs have been used; however, similar approaches for canine MSCs are relatively unexplored. Accordingly, we evaluated immunomodulatory effects and mechanisms in canine MSCs treated with TNF-α and IFN-γ. Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were incubated with the conditioned media (CM) from canine MSCs for 48 h. Expression of RNA was assessed by quantitative reverse transcription PCR (qRT-PCR), and protein levels were assessed by western blot. Expression of inducible nitric oxide synthase (iNOS), IL-6 and IL-1β was significantly (one-way ANOVA) decreased in LPS-stimulated RAW 264.7 cells incubated with CM from canine MSCs compared to that in LPS-stimulated RAW 264.7 cells alone. Furthermore, anti-inflammatory effects of TNF-α- and IFN-γ-primed canine MSCs were significantly increased compared with those of naïve canine MSCs. Expression of cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) were likewise significantly increased in primed canine MSCs. The level of iNOS protein in LPS-stimulated RAW 264.7 cells incubated with CM from the primed canine MSCs was decreased, but it increased when the cells were treated with NS-398(PGE2 inhibitor). In conclusion, compared with naïve canine MSCs, cells primed with TNF-α and IFN-γ cause a greater reduction in release of anti-inflammatory cytokines from LPS-stimulated RAW 264.7 cells; the mechanism is upregulation of the COX-2/PGE2 pathway.