Improvement plans on the operation of the Institutional Animal Care and Use Committee: focusing on the case of Seoul National University

Background : The Institutional Animal Care and Use Committee (IACUC) became compulsory in 2008 by the Animal Protection Act in Korea. Seoul National University (SNU), which conducts 5% of Korea’s total animal protocol reviews and uses 10% of national laboratory animal usage, has been influential in the review of animal protocols and management of animal facilities. This study was undertaken to suggest the operational improvement of the IACUC. It focused on the case of SNU. Results : The methodological framework consists of a qualitative approach. In particular, this study is focused on the grounded theory approach and sixty people were surveyed through purposeful sampling. Through this study, we found that various practical educations are necessary such as: (1) education for researchers on how to write a protocol, (2) standardization of screening criteria for various animal experiments by presenting various cases, (3) training on a detailed understanding of relevant laws and policies. In particular, an integrated management system, making it possible to share information among the related committees, would be essential for smoother operation of the IACUC. Conclusions : If various levels of education and the integrated management system are established, it will be possible to enhance the excellence of researchers and to better manage the operation of the IACUC.This study was supported by the Research Afair of SNU (2021 Policy Research Fund to Roh S)

A Rho Kinase (ROCK) Inhibitor, Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.Y

Osteogenic Differentiation Potential in Parthenogenetic Murine Embryonic Stem Cells

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and ß-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway

Abstract Background Bone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms. Methods hDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria. Results The calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture. Conclusions The stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration

Nuclear transfer by Using a Laser-Assisted Zona Pellucida Piercing Technique in Mice

Somatic cells nuclear transfer (SCNT) is a useful tool in studies of developmental biology and animal cloning. However, SCNT experiments only are allowed to skilled technical experts. In this experiment, laser-assisted zona pellucida piercing tool (LASER) was applied in murine SCNT. LASER minimized the use of piezo-driven micromanipulator (PIEZO), reducing chances of problems caused by PIEZO pulses. LASER reduced time that took to pierce zona pellucida in removal of nucleus from oocyte and somatic cell injection, which might have taken longer time with PIEZO. Time and difficulties that took researcher of equivalent skilled for their experiments were decreased with LASER, and this might affect the improvement of embryonic development. (LASER, 6.2% versus PIEZO, 2.9%; P＜ 0.05). Thus, these data support that the use of LASER can be used for zona pellucida piercing in murine SCNT program as an alternative to PIEZO

Birth of rats following nuclear exchange at the 2-cell stage

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced

In Vitro Development of Interspecies Nuclear Transfer Embryos: Effects of Culture Systems

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p<0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-cell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro

Effect of the timing of oocyte activation on development of rat somatic cell nuclear transfer embryos

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM SrCl₂ for 4 h (immediate activation after injection; IAI), or cultured in vitro for 2~3 h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation 2~3 h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of 36.6%(15/41), 39.5% (17/43) and 46.3% (25/54), respectively. However, in the ABI group, only one embryo (1.8%, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage (4.9%, 2/41). However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.This study was supported by Seoul National University Foundation (850-20030102

Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs