Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

BACKGROUND AND AIMS: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. METHODS: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). RESULTS: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. CONCLUSIONS: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album.

INTRODUCTION: The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties. OBJECTIVE: This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10-1, 1×10-2 potency (VAD1D2). METHODS: MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test. RESULTS: At the concentration of 10 µL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 µL/mL presented absolute percentages of initial apoptosis twice as high as at 10 µL/mL, which was similar to the control (untreated group). CONCLUSION: The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.

Canine atopic dermatitis attenuated by mesenchymal stem cells.

OBJECTIVE: To evaluate the use of mesenchymal stem cells (MSCs) in the attenuation of canine atopic dermatitis (AD). MATERIALS AND METHODS: Sixteen dogs were selected and divided into three groups, mild, moderate, and severe, according to the Canine Atopic Dermatitis Extent and Severity Index (CADESI-4). They were evaluated for 82 days. The protocol recommended in this experiment was to inject 2 × 106/kg bodyweight of MSC's in all groups by the intravenous route with intervals of applications of 21 days. The degree of pruritus was evaluated by examining the visual analog scale, the CADESI-4, the histopathology of the skin, hematological and biochemical parameters, the pyogenic effect of MSCs, and the thickness of the epidermis. RESULTS: There was a significant difference in the reduction of epidermal thickness in the moderate and severe groups. Hematological, biochemical, and body temperature parameters remained within normal limits for the species with no side effects. CONCLUSION: MSCs attenuated the clinical signs of AD.

Homoeopathic Viscum album (10-3) is More Cytotoxic to in vitro Culture of Human Breast Cancer Cell Line than to Human Mesenchymal Stem Cells

BackgroundBreast cancer has been considered a public health problem and homeopathic treatments are becoming increasingly recommended due to its ways of action and absence of adverse effects. MCF-7 is an adenocarcinoma of human breast cell line useful as preclinicalmodel to screen therapeutic agents such as ultra-diluted Viscum album, an European plant which extract is commonly used in cancer therapy. AIMS MCF-7 and mesenchymal stem cells (MSC) were used to evaluate the in vitrocytotoxicity of homoeopathic Viscum album 1x10-3(VA3X). Methodscells were cultured for 24 hours in controlled environment (37.5oC and 5% CO2) in 96-well plates. After this time, VA3X was added to the culture medium in concentrations varying from 10 to 100 L/mL.A control group was maintained with culture medium only. Cells were cultivated for 48 hours in these conditions for evaluation of cell viability by MTT assay. ResultsHigher cytotoxicity was observed in MCF-7 when compared to MSC, as the lower concentration of VA3X was capable of inducing tumor cell death and not healthy cell death. The MTT assay results were that 42 L/mL of VA3X reduced MCF-7 cells viability to 50% and 62 L/mL reduced MSC cells to the same percentage, what means that tumor cells are more sensible to VA3X than heathy cells. ConclusionViscum albumpresented higher cytotoxic action on human breast cancer cell line culture than on mesenchymal stem cells. This medicine is extensively used against cancer, and the use of the homoeopathic form of it brings new possibilities as no or fewer adverse effects would be present.(AU)