CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice / 한국실험동물학회지

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice / 한국실험동물학회지

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47(−/−) hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47(−/−) mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47(−/−) mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47(−/−) mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47(−/−) mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47(−/−) mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47(−/−) mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

Preclinical safety assessment of Angelica acutiloba using a 13-week repeated dose oral toxicity study in rats / 한국실험동물학회지

Angelica acutiloba (AA), a Japanese species of Danggui, has been used worldwide as a traditional herbal medicine with several bioactivities including anti-diabetic, anti-allergic, anti-inflammatory, anti-tumor, and anti-obesity. However, there is lack of toxicological data available to evaluate potential long-term toxicity and the no-observed-adverse-effect level (NOAEL) of AA extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In the 14-day repeat-dose toxicity study, no adverse effects on mortality, body weight change, clinical signs, and organ weights was found following repeat oral administration to rats for 14 days (125, 250, 500, 1000, and 2000 mg/kg body weight), leading that 2000 mg/kg is the highest recommended dose of AA extract for the 13-week repeat-dose oral toxicity study. In the 13-week repeat-dose oral toxicity study, the AA extract was orally administered to groups of rats for 13 weeks (125, 250, 500, 1000, and 2000 mg/kg body weight) to compare between control and AA extract groups. The administration of AA extract did not produce mortality or remarkable clinical signs during this 13-week study. And, the data revealed that there were no significant differences in food/water consumption, body weight, hematological parameters, clinical chemistry parameters, gross macroscopic findings, organ weight and histopathology in comparison to the control group. On the basis of these results, the subchronic NOAEL of the AA extract was more than 2000 mg/kg/day when tested in rats. And, the AA extract is considered safe to use orally as a traditional herbal medicine.

Evaluation of in vitro and in vivo genotoxicity of Angelica acutiloba in a standard battery of assays / 한국실험동물학회지

Among three representative species of Angelica found in Asian countries, including Korea, China, and Japan, Angelica acutiloba (AA) has been used as traditional herbal medicine with antitumor, anti-inflammatory, anti-obesity, and anti-diabetes activities. In this study, the potential genotoxicity and mutagenicity of the AA extract were examined in a battery of in vitro and in vivo tests (bacterial reverse mutation assay, in vitro chromosomal aberrations assay, and in vivo micronucleus assay) in accordance with the test guidelines for toxicity testing developed by the Organization for Economic Cooperation and Development. Upon testing in the bacterial mutation assay (Ames test) using five Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537, no significant increase the number of revertant colonies in the metabolic activation system and non-activation system was noted in the AA extract groups. Also, in the chromosome aberration test, the AA extract did not cause chromosomal aberration with or without metabolic activation by S9 mix. A bone marrow micronucleus test of mice demonstrated that the incidence of micronucleated polychromatic erythrocytes in the AA extract groups (500, 1000 and 2000 mg/kg BW) was equivalent to that of the negative control group. Based on these results from a standard battery of assays, the AA extract was concluded to have no genotoxic at the proper dose.

Gold thread implantation promotes hair growth in human and mice / 한국실험동물학회지

Thread-embedding therapy has been widely applied for cosmetic purposes such as wrinkle reduction and skin tightening. Particularly, gold thread was reported to support connective tissue regeneration, but, its role in hair biology remains largely unknown due to lack of investigation. When we implanted gold thread and Happy Lift™ in human patient for facial lifting, we unexpectedly found an increase of hair regrowth in spite of no use of hair growth medications. When embedded into the depilated dorsal skin of mice, gold thread or polyglycolic acid (PGA) thread, similarly to 5% minoxidil, significantly increased the number of hair follicles on day 14 after implantation. And, hair re-growth promotion in the gold threadimplanted mice were significantly higher than that in PGA thread group on day 11 after depilation. In particular, the skin tissue of gold thread-implanted mice showed stronger PCNA staining and higher collagen density compared with control mice. These results indicate that gold thread implantation can be an effective way to promote hair re-growth although further confirmatory study is needed for more information on therapeutic mechanisms and long-term safety.

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.

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 Human Fibroblasts in Mice: Tumorigenicity, 13-week Toxicity and Distribution Studies / 한국실험동물학회지

Human fibroblasts were developed for cellular therapy with the aim of correcting of depressed scars, but the safety of that in vivo is unclear. In this study, we assessed the safety of human fibroblasts by investigating the tumorigenicity, 13-week toxicity and through distribution studies. In the tumorigenicity test, nude mice were divided into three dosage level treatment groups with a negative/positive control group. At 6 months after intradermal transplantation, all of the treatment groups showed no development of a nodule on the injection sites and organs. Toxicity studies were performed using ICR and BALB/c mice for 13 weeks. The mice were divided into three dosage level treatment groups with a control and a syngeneic group. There was no treatment-related effect on clinical signs, mortality, body weight, food/water consumption, hematology, serum biochemistry, urine, necropsy findings and histopathological findings in any groups. These results suggest that the no-observed-effect level (NOEL) of the human fibroblasts was greater than 7.5x10(7) cells/kg for mice. In the distribution study, groups were treated with fibroblasts labeled with a fluorescent dye (CM-DiI) at low and high doses with a control and a syngeneic group. At 24 hours, a large percentage of the labeled fibroblasts were observed at the dermal layer. At 3 months, fluorescence of the labeled fibroblasts continued to be observed. Other tissues were not detected the fluorescence at any time. These studies demonstrate that the safety of human fibroblasts is reasonable with no toxic effect, no tumorigenicity and retention in the dermis. Our studies define preclinical safety testing standards relevant to the development of cellular therapeutics.