Natural killer (NK) cells have cytotoxic effects on tumor cells and viral pathogens. NK cell-derived exosomes (NK-exosomes) also express typical NK cell markers and cytotoxic molecules, therefore, exert anti-tumor and immune homeostatic activities. In this study, canine NK-exosomes separated from cytotoxic NK cell supernatant carried specific markers such as CD81, Alix, and Perforin 1. We examined the anti-tumor effects of NK-exosomes in an experimental murine model using the canine mammary carcinoma cells, REM134. REM134 cells were xenografted of mammary fat pad of mice. CD133, Bmi-1, MMP-3, IL-6, TNF-α, and PCNA are useful as a molecular marker for tumorigenesis and metastasis. The treatment of canine NK-exosomes inhibited tumor growth and significantly (p<0.01) downregulated the expression of Bmi-1, MMP-3, IL-6, TNF-α, and PCNA in REM134-treated mice. Also, the expression of CD133, potent cancer stem cell marker, was significantly downregulated in the canine NK-exosomes-treated mice compared with that of the tumor group. Collectively, these results suggested that canine NK-exosomes has a potential capacity for regulation of cancer progression and metastasis against canine mammary carcinoma.

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and thus hold therapeutic implications in field of regenerative medicine and reproductive biotechnology. In the present study, we compared the stem cell properties of bovine ear skin tissue (ESK)- and nasal mucosa (NM)-derived MSCs. Bovine ESK-MSCs and NM-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity during the 20 consecutive passages. Both ESK-MSCs and NM-MSCs showed similar morphology and expressed common cell surface markers (CD29, CD44, CD90, and CD105). Also, we compared differentiation potentials of bovine ESK-MSCs and NM-MSCs into osteogenic, adipogenic, and chondrogenic lineages through specific staining and quantitative real-time RT-PCR. As results, bovine ESK-MSCs and NM-MSCs could differentiate into mesodermal cell lineages. However, bovine ESK-MSCs and NM-MSCs exhibited difference in expression of differentiation-related specific markers. Specifically, NM-MSCs exhibited increased expression levels of osteocalcin, peroxisome proliferator-activated receptor gamma, and aggrecan compared to ESK-MSCs. Also, ESK-MSCs exhibited increased expression levels of collagen type I, II, and lipoprotein lipase compared to NM-MSCs. We suggest that the nasal mucosa of bovine could be used as a source of bovine MSCs.

Canine mammary tumors account for ~30% of all tumors in the female dogs and approximately 50% of the tumors are malignant. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. In this study, we examined the anti-tumor effects of canine mesenchymal stem cells-derived exosomes (MSC-exosomes) in an experimental murine mammary tumor model using canine mammary carcinoma cells, REM134. The MSC-exosomes were injected tumor site and tail vein of REM134 xenografted mice. We found that tumor size of the MSC-exosomes-treated group decreased compared to those of the only tumor group in REM134-driven tumorigenic mouse model. In addition, the MSC-exosomes-treated tumor group showed meaningfully reduced expression levels of the MMP-3, IL-1β, IL-6, and TNF-α compared to those in the tumor group. Specifically, we confirmed that the expression level of the CD133, potent cancer stem cell (CSC) markers, decreased in the MSC-exosomes-treated tumor group compared to the tumor group. This study suggests that the MSC-exosomes exhibited anti-tumor effects through downregulating CSC-related markers in the canine mammary tumor murine model. Further study is needed in the future, and we are conducting research on the detailed anti-tumor mechanism of the MSC-exosomes.

Porcine parvovirus 2 (PPV2) was recently detected in the Republic of Korea. This paper reports two near-complete genome sequences of PPV2 identified for the first time in the lung tissue of aborted pig fetuses.

The culture of the intestinal epithelium into three dimensional (3D) structures typically termed organoid culture. Organoid culture is based on the ability of intestinal stem cells (ISCs), at the base of the crypt, perpetually to divide and produce a fully differentiated, polarized epithelium. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) positive ISCs isolated from the intestine can form organoids in long-term culture. Thus, when cultured under the appropriate 3D conditions, single Lgr5+ ISCs undergo cycles of self-renewal, differentiation and morphogenesis, and self-organize into crypt-villus domains that house cycling ISCs and differentiated intestinal epithelial cells (IECs). In this study, we performed isolation, characterization and consecutive subculture of small intestinal crypts from BALB/c-nude mouse. Briefly, isolated mouse crypts were embedded in matrigel, cast into 40 μL droplets at the bottom of well in a 48-well plate. Following polymerization, the gels were overlaid with ISCs expansion medium containing B27, N2, N-acetylcysteine, epidermal growth factor, noggin, and R-spondin 1. As a result, mouse crypt-derived ISCs had enteroids and spheroid morphologies. We also confirmed by quantitative real-time RT-PCR that expression of ISCs-related specific genes (Lgr5, sox9) and IECs-related specific genes (chromogranin A, defensin-5, mucin-1, mucin-2, and villin) was maintained at eight passages or more. Thus, we observed that expression of specific markers and consecutive self-renewing in the mouse small intestinal crypt-derived organoids.

Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.

Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. Use of low-frequency ES for therapeutic purposes has been increasing during the last decades. Mesenchymal stem cells (MSCs) represent an appealing alternative cell source for cartilage repair. There are studies that induce differentiation into cartilage cells by treating the growth factors in stem cells or altering the properties of stem cells by genetic modification. In this study, we exposed equine adipose tissue-derived MSCs (eAD-MSCs) to ES and assessed changes in the chondrogenic differentiation potential. The cells obtained from equine adipose tissue attached to culture plates and expanded in vitro. Flow cytometric analysis at third passage indicated that the cells were strongly positive for CD44, CD90, and CD105, but negative for CD13, CD34, and CD45. Next, ES was applied to eAD-MSCs cultured under condition of high-density micromass under ES of 10 V/cm, with duration of 10 ms and a frequency of 2.0 Hz for three days. Gene expression of chondrogenic markers such as collagen type II, Aggrecan, and Sox9 was analyzed at three days of ES. As a result, we observed the differentiation potential of eAD-MSCs into chondrocytes by specific ES in absence of exogenous growth factors. We also found that ES upregulated the expression of heat shock protein 70, which affects cartilage formation. This study may contribute to the differentiation of MSCs into chondrogenic lineage under specific ES condition.

Mesenchymal stem cells (MSCs) are capable of differentiating into mesenchymal tissue such as bone, cartilage, muscle, and adipose, and have been isolated and characterized from various species. Deer adipose tissue-derived MSCs (dAD-MSCs) have not been studied and deer bone marrow-derived MSCs (dBM-MSCs) have not been fully characterized. In this study, we firstly isolated MSCs from deer tissues and then compared characteristics of dAD-MSCs and dBM-MSCs. dAD-MSCs and dBM-MSCs exhibited significant increase in proliferation under low-glucose DMEM culture condition during 20 and 10 passages consecutive passages, respectively. Both cells expressed cell surface markers such as CD73, CD90, and CD105, but did not express CD34 and CD45. Two types of cells expressed stemness markers (Oct4, Sox2, and Nanog) and exhibited differentiation potential into mesodermal lineages. Both cells exhibited osteogenic and chondrogenic differentiation potential, but poor adipogenic differentiation potential. Specifically, dAD-MSCs have a greater capacity for chondrogenic differentiation potential compared to dBM-MSCs. Collectively, we successfully isolated dAD-MSCs from deer for the first time. This study suggests that adipose tissue of deer could be used as a source of deer MSCs.

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesodermal lineages. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. Goat MSCs isolated from bone marrow (BM) or adipose tissue (AD) should be evaluated using in vitro assays, prior to their application in a tissue engineering project. In this study, we compared the stem cell properties of MSCs derived from goat AD, BM and ear skin tissue (ESK). As results, BM and ESK-MSCs exhibited a spindle-shaped morphology comparable to that of AD-MSCs. Especially, BM-MSCs could be cultured for significantly longer periods and exhibited the greatest expansion capacity, whereas AD-MSCs had the shortest culture time and lowest growth rate. Also, we compared differentiation potentials of AD, BM and ESK-MSCs into adipogenic, chondrogenic, and osteogenic lineages through specific staining and quantitative real-time RT-PCR. Collectively, we successfully isolated ESK-MSCs from goat for the first time. This study suggests that adult skin tissue of goat could be used as a source of goat MSCs. Further studies are needed to show the more information for establishment and fully characterization of goat ESK-MSCs.

Rabies is a zoonotic disease that causes severe destruction to the central nerve system which is usually fatal. It is one of the most important disease around the world and particular in Asia because of the high costs of prevention and post-exposure treatment. After the recurrence of sylvatic rabies in 1993, the number of raccoon dog mediating rabies cases in Korea has maintained annually until 2011. To better understand the current rabies epidemics in Korea, Korean rabies isolate (SKRBV0601GY) from Gyeonggi province in 2006 was compared with previous isolates in Korea and with isolates originating from the North-East Asia, such as Japan, China and Russia, based on complete nucleoprotein (N) gene sequences. By comparison of the N genes among these viruses, SKRBV0601GY revealed that nucleotide similarity ranged from 97.7 to 99.7%, 96.4 to 97.5%, 91.4 to 96.3%, 89.2 to 90% and 86.1 to 88.1% with Korean isolates, "Arctic-like-2" viruses, "Arctic" viruses, Russian group C - E and Chinese isolates, respectively. Phylogenetic analyses of the isolates revealed that the Korean isolate in 2006 belonged to Korean group B. The topology of the phylogenetic tree of Korean isolates related not the species and year of isolation but the geological location of the virus isolates. All of the Korean isolates showed close relationship to the "Arctic-like-2" virus (Russian group B) more than the "Arctic" virus (Russian group A) and all of the Chinese isolates (Chinese group A, B and C). The "Arctic-like-2" virus group contains the Japanese isolate and Russian group B viruses, originating from the south of East Siberia and Far East in Russia. These molecular data demonstrated that the current rabies epizootic in Korea developed independently of Chinese groups and originated from the "arctic-like-2" viruses in detail.