Euna Kwon

Safety of Intravenous Infusion of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Animals and Humans

Adipose tissue-derived mesenchymal stem cells (AdMSCs) represent an attractive and ethical cell source for stem cell therapy. With the recent demonstration of MSC homing properties, intravenous applications of MSCs to cell-damaged diseases have increased. In the present study, the toxicity and tumorigenicity of human AdMSCs (hAdMSCs) were investigated for clinical application. Culture-expanded hAdMSCs showed the typical appearance, immunophenotype, and differentiation capacity of MSCs, and were genetically stable at least 12 passages in culture. Cells suspended in physiological saline maintained their MSC properties in a cold storage condition for at least 3 days. To test the toxicity of hAdMSCs, different doses of hAdMSCs were injected intravenously into immunodeficient mice, and the mice were observed for 13 weeks. Even at the highest cell dose (2.5×10(8) cells/kg body weight), the SCID mice were viable and had no side effects. A tumorigenicity test was performed in Balb/c-nu nude mice for 26 weeks. Even at the highest cell dose (2×10(8) MSCs/kg), no evidence of tumor development was found. In a human clinical trial, 8 male patients who had suffered a spinal cord injury >12 months previous were intravenously administered autologous hAdMSCs (4×10(8) cells) one time. None of the patients developed any serious adverse events related to hAdMSC transplantation during the 3-month follow-up. In conclusion, the systemic transplantation of hAdMSCs appears to be safe and does not induce tumor development.

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.

Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats

As a well-known traditional medicine, Angelica gigas (AG) and its active constituents, including decursin and decursinol, have been shown to possess several health beneficial properties such as anti-bacterial, immunostimulating, anti-tumor, neuroprotective, anti-nociceptive and anti-amnestic activities. However, there is lack of toxicity studies to assess potential toxicological concerns, especially long-term toxicity and genotoxicity, regarding the AG extract. Therefore, the safety of AG extract was assessed in subchronic toxicity and genotoxicity assays in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a subchronic toxicity study for 13 weeks (125, 250, 500, 1000 and 2000 mg/kg body weight, delivered by gavage), data revealed no significant adverse effects of the AG extract in food consumption, body weight, mortality, hematology, biochemistry, necropsy, organ weight and histopathology throughout the study in male and female rats. These results suggest that no observed adverse effect level of the AG extract administered orally was determined to be greater than 2000 mg/kg/day, the highest dose tested. In addition, a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay suggested that the AG extract was not genotoxic. In conclusion, the AG extract appears to be safe as a traditional medicine for oral consumption.

Evaluation of subchronic (13week) toxicity and genotoxicity potential of vinegar-processed Genkwa Flos.

Genkwa Flos (GF) is a well-known traditional medicine that is used to treat tumors and to relieve inflammation-related symptoms. GF tends to be taken in repeated doses for a long period of time, and although many reports on the toxicity of raw GF have led to a processing method to remove the toxicity, little information is currently available with regards to the toxic effects of subchronic exposure to processed GF (PGF). The aim of this study was to assess the possible genotoxicity and subchronic toxicity of PGF extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study was carried out with rats, and the change in body weight observed in rats receiving PGF extract was normal. It is worth noting that the PGF extract groups exhibited an obvious increase in liver weight along with a significant increase in serum alkaline phosphatase activity at doses of 667 and 2000mg/kg, providing evidence of hepatotoxic potential. More importantly, the results of the Ames test indicated that the PGF extract presented a mutagenic potential. Altogether, these results are the first to determine the subchronic toxicity and genotoxicity of the PGF extract, indicating that when GF is used for medicinal purposes, the period of use should be considered despite the manner in which the extract is processed.

Antiangiogenic effect of betaine on pathologic retinal neovascularization via suppression of reactive oxygen species mediated vascular endothelial growth factor signaling.

Reactive oxygen species (ROS) as well as vascular endothelial growth factor (VEGF) play important roles in pathologic retinal neovascularization. We investigated whether betaine inhibits pathologic retinal neovascularization in a mouse model of oxygen induced retinopathy (OIR). Betaine was intravitreally injected in OIR mice at postnatal day (P) 14. At P17, the neovascular tufts area in OIR retina was analyzed. Intravitreal injection of betaine (200μM) effectively reduced the neovascular tufts area in OIR retina (68.0±6.7% of the control eyes, P<0.05). Even in a high concentration (2mM), betaine never induced any retinal toxicity or cytotoxicity. Betaine significantly inhibited VEGF-induced proliferation, migration, and tube formation in human retinal microvascular endothelial cells (HRMECs). Betaine suppressed VEGF-induced VEGFR-2, Akt and ERK phosphorylation in HRMECs. In human brain astrocytes, betaine reduced tBH-induced ROS production, and subsequently attenuated tBH-induced VEGFA mRNA transcription via suppression of ROS. Our data suggest that betaine has an anti-angiogenic effect on pathologic retinal neovascularization via suppression of ROS mediated VEGF signaling. Betaine could be a potent anti-angiogenic agent to treat pathologic retinal neovascularization.

Pre-clinical in vitro and in vivo safety evaluation of Cimicifuga heracleifolia

The rhizomes of Cimicifuga species, including Cimicifuga heracleifolia (CH), have been widely used as antipyretic, analgesic, and anti-inflammatory agents in oriental countries. However, information regarding its toxicity, especially long-term toxicity and genotoxicity, is limited. Therefore, we performed the subchronic toxicity and genotoxicity assays of the CH extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a 13-week repeat-dose oral toxicity study, the CH extract did not influence body weight, food/water consumption, mortality, clinical signs, and urinalysis throughout the study. Noteworthy, the CH extract groups exhibited increased liver weights along with serum alanine transaminase activity rise at doses of 667 and 2000 mg/kg in females. No-observed-adverse-effect-level of the CH extract administered orally was concluded to be 2000 mg/kg body weight/day for male rats and 222 mg/kg body weight/day for female rats. The CH extract did not exert a mutagenic or clastogenic effect in Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. Overall findings of the subchronic toxicity study indicate for the first time that the CH extract may possess hepatotoxic potential in female rats, suggesting that further mechanistic studies should be performed to have more conclusive results on hepatotoxic potential of the CH extract.

Acute and subchronic toxicity of FCD, a soybean extract combined with l-carnitine, in Sprague–Dawley rats

Soy products are primarily composed of proteins, phytochemicals such as isoflavones, soy lipids, and carbohydrates. Recently, soy isoflavones with L-carnitine were reported to exhibit anti-obesity effects in mice. FCD, a combination of soybean extract and L-carnitine, is a newly developed food substance. As a part of its safety assessment, acute and 13-week subchronic toxicity studies were performed in a total of 100 Sprague-Dawley (SD) rats. In the acute study, a single limit dose of 2000 mg/kg was orally administered to five male and five female rats. No adverse effects or mortality was observed during a 14-day period or upon gross pathological examination. In the subchronic study, FCD was orally administered in daily doses of 500, 1000, and 2000 mg/kg for 13 weeks, resulting in no mortality, and no changes in hematological and serum biochemistry parameters, gross pathology or histopathology. However, body weights of females were significantly decreased 10 weeks after treatment at an average of 2000 mg/kg. In addition, a slight decrease in mean food and water consumption was observed at the same dose level for 13 weeks. Therefore, the no-observed-adverse-effect-level (NOAEL) of FCD was considered to be 2000 mg/kg for male and 1000 mg/kg for female SD rats.

Neurometabolic profiles of the substantia nigra and striatum of MPTP-intoxicated common marmosets: An in vivo proton MRS study at 9.4 T.

Given the strong coupling between the substantia nigra (SN) and striatum (STR) in the early stage of Parkinsons disease (PD), yet only a few studies reported to date that have simultaneously investigated the neurochemistry of these two brain regions in vivo, we performed longitudinal metabolic profiling in the SN and STR of 1‐methyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐intoxicated common marmoset monkey models of PD (n = 10) by using proton MRS (1H–MRS) at 9.4 T. T2 relaxometry was also performed in the SN by using MRI. Data were classified into control, MPTP_2weeks, and MPTP_6‐10 weeks groups according to the treatment duration. In the SN, T2 of the MPTP_6‐10 weeks group was lower than that of the control group (44.33 ± 1.75 versus 47.21 ± 2.47 ms, p < 0.05). The N‐acetylaspartate to total creatine ratio (NAA/tCr) and γ‐aminobutyric acid to tCr ratio (GABA/tCr) of the MPTP_6‐10 weeks group were lower than those of the control group (0.41 ± 0.04 versus 0.54 ± 0.08 (p < 0.01) and 0.19 ± 0.03 versus 0.30 ± 0.09 (p < 0.05), respectively). The glutathione to tCr ratio (GSH/tCr) was correlated with T2 for the MPTP_6‐10 weeks group (r = 0.83, p = 0.04). In the STR, however, GABA/tCr of the MPTP_6‐10 weeks group was higher than that of the control group (0.25 ± 0.10 versus 0.16 ± 0.05, p < 0.05). These findings may be an in vivo depiction of the altered basal ganglion circuit in PD brain resulting from the degeneration of nigral dopaminergic neurons and disruption of nigrostriatal dopaminergic projections. Given the important role of non‐human primates in translational studies, our findings provide better understanding of the complicated evolution of PD.

Behavior, PET and histology in novel regimen of MPTP marmoset model of Parkinson’s disease for long-term stem cell therapy

Stem cell technologies are particularly attractive in Parkinson’s disease (PD) research although they occasionally need long-term treatment for anti-parkinsonian activity. Unfortunately, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) widely used as a model for PD has several limitations, including the risk of dose-dependent mortality and the difficulty of maintenance of PD symptoms during the whole experiment period. Therefore, we tested if our novel MPTP regimen protocol (2 mg/kg for 2 consecutive days and 1 mg/kg for next 3 consecutive days) can be maintained stable parkinsonism without mortality for long-term stem cell therapy. For this, we used small-bodied common marmoset monkeys (Callithrix jacchus) among several nonhuman primates showing high anatomical, functional, and behavioral similarities to humans. Along with no mortality, the behavioral changes involved in PD symptoms were maintained for 32 weeks. Also, the loss of jumping ability of the MPTP-treated marmosets in the Tower test was not recovered by 32 weeks. Positron emission tomography (PET) analysis revealed that remarkable decreases of bindings of 18F-FP-CIT were observed at the striatum of the brains of the marmosets received MPTP during the full period of the experiment for 32 weeks. In the substantia nigra of the marmosets, the loss of tyrosine hydroxylase (TH) immunoreactivity was also observed at 32 weeks following the MPTP treatment. In conclusion, our low-dose MPTP regimen protocol was found to be stable parkinsonism without mortality as evidenced by behavior, PET, and TH immunohistochemistry. This result will be useful for evaluation of possible long-term stem cell therapy for anti-parkinsonian activity.

Dual effects of human adipose tissue-derived mesenchymal stem cells in human lung adenocarcinoma A549 xenografts and colorectal adenocarcinoma HT-29 xenografts in mice

Human adipose tissue-derived mesenchymal stem cells (hATMSCs) have great potential as a therapy for various diseases. However, emerging evidence shows that there are conflicting results concerning effects of hATMSCs on tumor progression. Our objective was to determine whether and how hATMSCs modulate tumor growth. After cancer cell lines were subcutaneously inoculated into BALB/c-nude and hairless severe combined immunodeficient mice, hATMSCs were intratumorally injected into the mice. The growth of the A549 tumors was inhibited by hATMSCs, yet that of the HT-29 tumors was significantly promoted by hATMSCs in the in vivo xenograft models. In vitro study using a co-culture system of cancer cells and hATMSCs was consistent with the in vivo experiments. To reveal the molecular events induced by hATMSCs in the xenograft models, global gene expression profiles of the A549 and HT-29 tumors in the absence or presence of hATMSCs were determined. Significant numbers of genes involved in biological processes were altered in the hATMSC-treated A549 tumors, whereas no biological process was regulated by treatment with hATMSCs in the HT-29 tumors, reflecting the different effects of hATMSCs in the different types of cancer. Notably, histone cluster 1, H2aj and neuropeptide Y receptor Y4 were found to be expressed in direct or inverse proportion to tumor size in both xenograft models. In addition, nuclear factor κB (NF-κB) p65 was differentially phosphorylated by the hATMSCs dependent on the source of the cancer cells. In conclusion, the identified gene profiling and NF-κB signaling provide molecular evidence to explain the conflicting findings in tumor‑MSC studies, although further study is needed to confirm these findings using various types of cancer.