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.

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.

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSCexo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 μg/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 µg/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) serve as a unique source for cell therapy. We investigated whether exosomes from iMSCs promote the proliferation of human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). iPSCs were established from human Wharton’s jelly MSCs and were allowed to differentiate into iMSCs. Exosomes were collected from the culture supernatant of MSCs (MSC-exo) and iMSCs (iMSC-exo), and their characteristics were investigated. Both exosome types possessed basic characteristics of exosomes and were taken up by skin cells in vitro and in vivo. A significant increase in HaCaT proliferation was observed with iMSC-exo, although both exosomes increased the viability and cell cycle progression in HaCaT and HDFs. No significant difference was observed in the closure of wound scratch and the expression of reparative genes between cells treated with the two exosome types. Both exosomes enhanced the secretion of collagen in HaCaT and HDFs; however, an increase in fibronectin level was observed only in HaCaT, and this effect was better with iMSC-exo treatment. Only iMSC-exo increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. Our results indicate that iMSC-exo promote the proliferation of skin cells by stimulating ERK1/2 and highlight the application of iMSCs for producing exosomes.

The objective of this study was to identify the proteins actively involved in the protection and repair of damaged cells, secreted by canine adipose derived mesenchymal stem cells (AT-MSCs) into the conditioned media. For this purpose, conditioned media (CM) was recovered from passage three stage canine AT-MSCs and skin fibroblasts cultured in serum free media after 24, 48 and 72 h. The extraction of exosomes was performed from 10-20 ml of CM using total exosome isolation kit. The isolated exosomes were then subjected to western analysis for the identification of annexin-I, annexin-II, histone H3 and dysferlin proteins. Results demonstrated the expression of proteins in the conditioned media isolated from canine AT-MSCs reflecting their potential in reducing the extent of damage at cellular levels. In conclusion, the conditioned media derived from canine AT-MSCs can be helpful in restoring the normal structure of cells both in vivo and in vitro conditions.