Protein and peptide candidates are screened to apply therapeutic application as a drug. Ensuring that these candidates are delivered and maximized effectiveness is still challenging and a variety of studies are ongoing. As drug delivery system vehicles, cell-penetrating peptide (CPP) can deliver various kinds of cargo into the cell cytosol. In a previous study, we developed Ara27 CPP, which are a zinc knuckle family protein of Arabidopsis, and confirmed internalization in human dermal fibroblasts and human dental pulp stem cells at low concentration with short time treatment condition without any toxicity. Ara27, an amphipathic CPP, could be modified and utilized in the biomedical field excluding the risk of toxicity. Therefore, we would like to confirm the non-toxic induced penetrating ability of Ara27 in various cell lines. The purpose of this study was to screen the cell internalization ability of Ara27 in various cell lines and to confirm Ara27 as a promising core CPP structure. First, Ara27 was screened to confirm non-toxicity concentration. Then, fluorescence-labeled Ara27 was treated on human normal cell lines, cancer cell lines and animal cell lines to identify the cellular internalization of Ara27. Ara27 was well intracellular localized in all cell lines and the intensity of fluorescence was remarkably increased in time pass manner. These results indicate that Ara27 has the potential as a core structure for applications in various drug delivery systems.

The use of animals heavily impacts the mental health of researchers performing the animal experiments. The animal researchers need to take care of animals but also give pain and sacrifice them at the same time. This circumstance can cause a variety of mental stress to the researchers. The stress generated in the laboratory would not only negatively affect the management of animals and the research results, but also would harm the researchers’ physical and mental health. Because the feeling of sympathy for animals is a natural feature of humanity, psychological stress following a laboratory animal’s death after use is not surprising. It is necessary to revise the relevant laws based on understanding the difficulties of animal researchers in society and to develop related educational programs at the national level to help the psychology and emotions of researchers who conduct animal experiments.

Salivary glands are exocrine glands that secrete saliva into the oral cavity, and secreted saliva plays essential roles in oral health. Therefore, maintaining the salivary glands in an intact state is required for proper production and secretion of saliva. To investigate a specific signaling pathway that might affect the maintenance of mouse submandibular gland (SMGs), RNA sequencing was performed. In SMGs, downregulated expression patterns of Rho-associated protein kinase (ROCK) signaling pathway-related genes, including Rhoa, Rhob, Rhoc, Rock1, and Rock2, were observed. Gene expression profiling analyses of these genes indicate that the ROCK signaling pathway is a potential signal for SMG maintenance.

Alopecia has emerged as one of the biggest interests in modern society. Many studies have focused on the treatment of alopecia, such as transplantation of hair follicles or inhibition of the androgen pathway. Hair growth is achieved through proper proliferation of the components such as keratinocytes and dermal papilla cells (DPCs), movement, and interaction between the two cells. The present study examined the effect of the hedgehog (Hh) signaling pathway, which is an important and fundamental signal in the cell, on the morphology and the viability of human keratinocytes and DPCs. Upregulation of Hh signaling caused a morphological change and an increase in epithelium-mesenchymal transition-related gene expression but reduced the viability of keratinocytes, while the alteration of Hh signaling did not cause any change in DPCs. The results show the possibility that the regulation of Hh signaling can be applied for the treatment of alopecia.

Hedgehog (Hh) pathway plays a key role in development from invertebrate to vertebrate. It is known to be involved in cell differentiation, polarity, proliferation, including the development of vertebrate limb and the establishment of flies’ body plan. To investigate how the regulation of Hh pathway affects the development of parthenogenetic murine embryos, the parthenogenetically activated murine embryos were treated with either cyclopamine (Cyc), an antagonist of Hh pathway, or purmorphamine, an agonist of Hh pathway. While Cyc did not affect the blastocyst formation and its total cell number, the chemical reduced the hatching rate of embryos and the expression levels of Fn1 mRNA. The results of the present study show the possibility that Cyc may affect the development of embryos at blastocyst stage by blocking Hh pathway and this may cause detrimental effect to the embryos at peri-, and post-implantation stages.

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus

Sonic hedgehog (Shh) signaling pathway plays a key role in the development of various vertebrate embryos and remains important in adults. Although Shh signaling pathway has widely been studied in post-implantation stage embryos, only few studies are reported about pre-implantation stage embryos. To investigate the effect of Shh on pre-implantation stage embryos, cyclopamine and purmorphamine were treated to embryos in culture. Cyclopamine acts as an antagonist of the hedgehog signaling because it has a high affinity to Smoothened, a key part of the hedgehog signaling pathway. On the other hand, purmorphamine activate Smoothened and acts as a Shh signaling agonist. The oocytes were collected after superovulation and parthenogenetically activated in Chatot, Ziomek, and Bavister medium (CZB) including 10 mM strontium for 5 hr. The activated oocytes were cultured in potassium simplex optimized medium (KSOM), KSOM with 5 uM of cyclopamine, KSOM with 1 uM of purmorphamine, or KSOM with both 5 uM of cyclopamine and 1 uM of purmorphamine. After 5.5 days in culture, there was no significant difference in blastocyst development among the four experimental groups. However, the hatching rate was increased in the groups containing purmorphamine, and the blastocysts of the purmorphamine-containing groups had higher total cell number than those of other two groups when the cells were counted after Hoechst33342 staining. Quantitative real-time PCR (qRT-PCR) shows the difference of gene expression level which are related to epithelial-mesenchymal transition (EMT). Taken together, this study suggests that the increase of Shh has an effect on the increases of EMT-related genes and hatching rate of pre-implantation stage embryos, and this may improve implantation subsequently.

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

Human malignant melanoma is an aggressive skin cancer which has been rising at a greater rate than any other cancers. Although various new therapeutic methods have been developed in previous studies, this disease has properties of high proliferation and metastasis rate which remain obstacles that have lead to a poor prognosis in patients. It has been reported that a specific Lactobacillus extract has anti-cancer and –metastasis effect in vitro and in vivo. However, previous research has not specified precisely what effect the Lactobacillus rhamnosus GG (LGG) extract has had on human malignant melanomas. In this study, we showed that the LGG extract has anti-cancer and –metastasis effects on the human malignant melanoma cell lines, A375P and A375SM. At first, it was found that, while the LGG extract affects human neonatal dermal fibroblasts slightly, it induced the dose-dependent anti-cancer effect on A375P and A375SM by a WST-1 proliferation assay. As a result of a real-time PCR analysis, the expression patterns of several genes related to cell cycle, proliferation, and apoptosis were modulating in a manner that inhibited the growth of both malignant melanoma cell lines after the treatment of the LGG extract. Furthermore, genes related to the epithelialmesenchymal transition were down-regulated, and migration rates were also decreased significantly by the LGG extract. Our study showed that the LGG extract could be used as a potential therapeutic source.

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher’s competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

Polo-like kinase 1 (plk1) shows multiple events of somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus at different stages of mitosis in spindle organization. Somatic cell nuclear transfer (SCNT) has a number of advantages however it is difficult to apply to basic or translational researches due to its low cloning efficiency. The causes of this low cloning efficiency are unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, a biological factor plk1 was investigated. B6D2F1 mice (7–8 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were then collected 14 hr after HCG injection and cultured on potassium simplex optimized medium (KSOM). The plk1-specific inhibitor BI2536 was used to understand the influence of plk1. The 2-cell stage embryos were assessed by fluorescence immunoassay. In consequence, all BI2536-treated embryos failed in the first mitotic division which showed plk1 have critical role in the first mitotic division of the mouse embryo. SCNT requires enucleation of oocyte and injecting a donor cell into the enucleated cytoplast. In this process, a respectable amount of plk1 that co-localize with nucleus may be removed together. Fluorescence immunoassay and qPCR were used to monitor the change of plk1 level during SCNT. There was significant difference between the control and enucleated embryos in the level of plk1. In all division-failure 2-cell embryos, incorrect positioning of plk1 was found. Taken together, this results demonstrate that plk1 is critical for successful mitotic division of mouse SCNT 1-cell embryos.

Although assisted reproductive technology (ART) has been developed in many mammalian species including cows, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to preserve embryos at room temperature. The basic medium for embryo preservation consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum (FBS). To maintain survival and prevent damage during embryo storage, three candidate small molecules were selected—CHIR99021, Y-27632 and Thiazovivin—and their concentrations were optimized. Then, the embryos in the small molecule supplemented preservation medium were stored at room temperature. The viability and hatching rate of embryos stored at 10°C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after storage at 20°C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The mechanism by which small molecules enhance survival of embryos during storage was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.

To have a better understanding of pluripotency, whole gene expression of embryo-derived stem cells (EdSCs) in bovine species was investigated. EdSCs were established from the embryos produced by in vitro fertilization, parthenogenesis and somatic cell nuclear transfer. Then, the microarray was performed and analyzed. Differently expressed genes (DEGs) were also confirmed by Real-time PCR. Among 10,203 DEGs, little difference was found in gene expression among three kinds of EdSCs. Conversely, all EdSCs have an immensely different gene expression when compared with somatic cells, consistent with scatter plat results. To investigate shared pathways for pluripotency in all EdSCs, 2,415 co-DEGs were identified which compared with somatic cells. By KEGG database, there were 54 signaling pathways in co-DEGs and some of them were related with pluripotency maintenance such as TGFβ, WNT and JAK-STAT signaling. In TGFβ signaling, BMP family and SMAD family were involved in co-up-regulated DEGs. In WNT signaling, WNT family and receptors were included in co-up-regulated DEGs, while inhibitors of WNT signaling were associated with co-down-regulated DEGs. In JAK-STAT signaling, STAT3 belonged to co-down-regulated DEGs. These DEGs were also confirmed by Real-time PCR. Taken together, BMP and WNT pathways may be activated and paly central roles to retain pluripotency in bovine EdSCs, whereas the LIF/STAT3 pathway may not be operated well. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and No. 2015048003 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea.

Skin-derived precursors (SKPs) have potential to differentiate to various cell types including osteoblasts, adipocytes and neurons. SKPs are a candidate for cell-based therapy since they are easily accessible and have multipotency. Most mammalian cells are exposed to a low oxygen environment with 1 to 5% O2 concentration in vivo, while 21% O2 concentration is common in in vitro culture. The difference between in vitro and in vivo O2 concentration may affect to the behavior of cultured cells. In this report, we investigated the effect of hypoxic condition on stemness and proliferation of SKPs. The results indicated that SKPs exposed to hypoxic condition for 5 days showed no change in proliferation. In terms of mRNA expression, hypoxia maintained expression of stemness markers; whereas, oncogenes, such as Klf4 and c-Myc, were downregulated, and the expression of Nestin, related to cancer migration, was also downregulated. Thus, SKPs cultured in hypoxia may reduce the risk of cancer in SKP cell-based therapy.

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3 hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine

Human dental pulp stem cells (DPSCs) are multi-potent mesenchymal stem cells that have several differentiation potentials. An understanding of thetissues that differentiate from these cells can provide insights for future regenerative therapeutics and tissue engineering strategies. The mesiodens is the most frequent form of supernumerary tooth from which DPSCs can differentiate into several lineages similar to cells from normal deciduous teeth. Recently, it has been shown that nanoscale structures can affect stem cell differentiation. In our presentstudy, we investigated the effects of a 250-nm nanoscale ridge/groove pattern array on the osteogenic and adipogenic differentiation of dental pulp cells from mesiodenscontaining human DPSCs. To this end, the expression of lineage specific markers after differentiation induction was analyzed by lineage specific staining and RT-PCR. The nanoscale pattern arrayed surface showed apositive effect on the adipogenic differentiation of DPSCs. There was no difference between nanoscale pattern arrayed surface and conventional surface groups onosteogenic differentiation. In conclusion, the nanoscale ridge/groove pattern arrayed surface can be used to enhance the adipogenic differentiation of DPSCs derived from mesiodens. This finding provides an improved understanding of the effects of topography on cell differentiation as well as the potential use of supernumerary tooth in regenerative dental medicine.

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest‐related precu- rsor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leuke- mia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our pre- sent study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT‐PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c‐Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog exp- ression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stem- ness in SKPs through the suppression of lineage differen- tiation and in part through the control of cell proliferation.

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of Tuj1 increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous Igf2 may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

In the first part of this study, a novel culture device the named oil-free micro tube culture (MTC) system for in vitro culture (IVC) of murine and porcine embryos was introduced. Parthenogenetic mouse and porcine embryos were placed into 0.2-mL thinwall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as the control. Murine embryos in MTC had a higher blastocyst formation rate and larger population of cells in the blastocysts. This was due to higher numbers of trophectoderm (TE) cells rather than inner cell mass cells. On the other hand, the 'MTC' system in the pig showed similar (in 20 μl medium volume) or lower (in 10 μl medium volume) blastocyst formation rate when compared with drop culture system. In the second part of this study, dexamethasone (DEX) and leukemia inhibitory factor (LIF), which suppress PGF2α, were directly supplemented into ET media, and transfer of the embryos to surrogate was followed. In the cattle industry, embryo transfer technology has been used to produce the most valuable cows or bulls. Numerous factors such as heat stress, mastitis, manipulating female reproductive tract may contribute to early embryonic loss through premature increases of uterine luminal concentrations of PGF2α in cows. Furthermore, addition of PGF2α to culture medium has been shown to inhibit the development and hatching of mammalian embryos. When DEX and LIF were supplemented, the pregnancy rate (6 month post-ET) was increased from 56.0% to 68.3%. In IVC experiment, DEX and LIF supplementation supported hatching of bovine embryos in the presence of PGF2α in the medium (from 16.9% to 40.6%). Additional ET experiments using alternative drugs are currently under investigation. The present work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF; 109020-3).