Effects of Different Concentrations of Reversine on Plasticity of Mesenchymal Stem Cells.

Dedifferentiation can be induced by small molecules. One of these small molecules used in this study in order to increase the plasticity of differentiation of stem cells was reversine. The objective of present study was to investigate the effect of different concentrations of reversine on the plasticity of ovine fetal bone-marrow mesenchymal stem cells (BM-MSCs). BM-MSCs were extracted from ovine fetal and cultured. Passaged-3 cells were evaluated for their differentiation potential into osteocytes and adipocytes cells. In the present study, BM-MSCs were culture plated in the presence of 0, 300, 600, 900 and 1200 nM of reversine. The number of viable cells was determined by MTT test after addition of different concentrations of reversine. Furthermore, expression of the nanog gene was evaluated. The culture without reversine was taken as the control group. Expression of nanog was analysed by immunocytochemistry. Multi-lineage differentiation showed that the BM-MSCs could be differentiated into adipose cells and osteocytes. Our results indicated that the addition of 1200 nM of reversine to medium significantly decreased overall proliferation compared to the other treatment groups (p > 0.05). Real-time PCR analysis showed that after addition of 600 nM of reversine significantly increased nanog expression compared to other treatments. All treatments received reversine were seen to be relative expression of nanog. Our findings confirm that low concentrations reversine increases the plasticity of ovine BM-MSCs.

Effects of irrigation with secondary treated wastewater on physicochemical and microbial properties of soil and produce safety in a semi-arid area.

Water scarcity is becoming one of the largest problems worldwide. Agricultural reuse of wastewater has been considered a valuable and reliable alternative, alleviating the pressure on freshwater resources in arid and semi-arid regions such as the Middle East. Inadequate microbial quality of treated wastewater is a challenge for developing countries, which limits agricultural reuse of wastewater. This study assessed the impact of irrigation with secondary treated wastewater (STWW) on soil properties as well as the safety of various types of crops as compared with tap water (TW) irrigation through a furrow system. Total and fecal coliforms and Escherichia coli were monitored as indicator bacteria in STWW, irrigated soil and harvested crops. The presence of pathogenic E. coli O157, Salmonella and Shigella was also monitored in all samples using a combination of culture and molecular methods. The microbial quality of wastewater in terms of E. coli concentration (4.18 Log MPN/100 ml) failed to meet the world health organization (WHO) recommendation for irrigation of root and leafy crops (≤103 and ≤ 104E. coli per 100 ml for root and leafy crops, respectively). No significant effects on physicochemical properties of the soil irrigated with STWW was found in comparison with control plots, except for electrical conductivity (EC) and sodium adsorption ratio (SAR), which were slightly higher in STWW soil samples. Although the microbial quality of soil was affected by STWW irrigation, a relatively low concentration of E. coli was detected in soil. No microbial contamination in terms of E. coli was found on harvested maize and onion. E. coli contamination of lettuce and spring onion was found for both irrigation schemes. No STWW, soil or crop samples were found positive for pathogenic bacteria. According to the analyzed parameters, STWW could be safely used as an alternative source for irrigation of root and leafy crops.

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

Ovine fetal mesenchymal stem cell differentiation to cardiomyocytes, effects of co-culture, role of small molecules; reversine and 5-azacytidine.

The aim of the present study was to investigate the effect of small molecules: Reversine and 5-azacytidine (5-AC), in an indirect co-culture condition with the cardiac fibroblasts as well as non co-culture condition, in order to explore the effect of such molecules in the process of differentiation of the ovine bone-marrow mesenchymal stem cells (BM-MSCs) towards cardiomyocytes. Surface antigens of the isolated cells were analysed using flow-cytometry. In addition, following to three passages cells were examined for their differentiation capacity into osteocytes and adipose cells, in order to ensure the mesenchymal origin of the stem cells. Six types of treatments were carried out in the present investigation, such that, in the first treatment BM-MSCs were cultured for 28 days as control group; the second treatment was composed of culturing ovine fetal cardiac fibroblasts on inserts, aiming to use these inserts for culturing plates which were seeded with BM-MSCs (Chamber group). As the third treatment, BM-MSCs were supplemented with 10-µM 5-AC and incubated for 48 h. The fourth treatment was composed of supplementing BM-MSCs with the 600-nM reversine, incubated for 48 h, and subsequently the incubation was further extended for another 48 h in the presence of 5-AC. The fifth treatment was composed of supplementing the chamber group with 10-µM 5-AC and incubation for 48 h, and the last or the sixth treatment was such that chamber group was supplemented with 600-nM reversine and an incubation period of 48 h. Following to the incubation, medium was replaced with 10-µM 5-AC and further incubated for another round of 48 h. In all treatments, following to addition of the small molecules incubations were carried out for 28 days; same as controls. Expression of cardiac alpha-actinin was analysed by immunocytochemistry. BM-MSCs have shown to express CD44 and CD166 along with a weak expression of the CD90, CD34, in addition to CD45. Multilineage differentiation has indicated that BM-MSCs could differentiate into adipose and osteocytes cells as well. In the treatment 4 it was observed that FGF signalling involved genes and all cardiac-related genes (ANP, MYH6 and Troponin I) were significantly expressed, except connexin 43 compared to other treatments. All treatments received small molecules, either alone or as a co-culture were seen to express sarcomeric alpha-actinin. This finding was partially supported by immunocytochemistry. These results validate that reversine and 5-AC have an effect on ovine BM-MSC differentiation into cardiomyocytes. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of feeding greater amounts of dietary energy for a short-term with or without eCG injection on reproductive performance, serum metabolites and hormones in ewes.

This study was conducted to compare the effect of transient high-energy diet in a short-term period with or without eCG injection on ovarian follicle development, twining rate, serum metabolites and hormones in ewes. A total of 45 estrous cyclic Naeini ewes were randomly assigned to three experimental groups: 1-Control (control), 2-High energy short-term feeding (HE), and 3-high energy short-term feeding + eCG injection (HEe). Ewes were housed in individual pens with free access to feed and water. The stage of the estrous cycle of all ewes was synchronized by insertion of intravaginal progesterone sponges. Focus feeding started from 4 days before until 1 day after sponge removal. Follicle development was monitored from 4 days before until 1 day after sponge removal and blood samples were taken during this time. Results showed that ewes fed high energy diets (HE and HEe) had greater (P < 0.05) large follicle numbers compared with the control group. Feeding high energy diets increased (P < 0.05) serum glucose, cholesterol and insulin, but had lesser (P < 0.05) serum urea nitrogen concentrations near the time of ovulation. After the start of experiment, ewes fed high energy diets had less (P < 0.05) serum estradiol. However, 1 day after sponge removal, serum estradiol in HE and HEe groups increased (P < 0.05). It was concluded that short-term (6-day) changes in amount of dietary energy with or without eCG injection increased twin births and had beneficial effects on the blood metabolites and hormone concentrations in Naeini ewes.

The interfering effects of superovulation and vitrification upon some important epigenetic biomarkers in mouse blastocyst.

Appropriate epigenetic changes in preimplantation embryos are critical for embryonic development and successful pregnancy. The aim of this study was to evaluate the effects of some assisted reproductive techniques (ARTs) on a panel of epigenetic biomarkers by immunofluorescence staining at blastocyst stage. For this purpose, four treatment groups were designed: control (C), superovulation (S), superovulation+in vitro culture (SI), and superovulation+vitrification+in vitro culture (SVI). Results showed that vitrification decreased the developmental competence of embryos cultured in vitro (P<0.05). Semi-quantitative analysis revealed that vitrification decreased the fluorescence intensity of global DNA methylation in the inner cell mass (ICM), in SVI Group in comparison to C group (P<0.05). Superovulation, elevated the level of H3K9acetylation of trophectoderm (TE) in comparison to C and SI groups (P<0.05). Furthermore, ARTs manipulations influenced H3K9acetylation in the ICM (P<0.05). The fluorescence intensity of H4K12acetylation in TE for SVI group was higher than C and S (P<0.05). For H3K4tri-methylation, S group had higher fluorescence intensity in the ICM in comparison to SI and SVI (P<0.05). Finally, in vitro culture decreased Pou5f1 protein signal in comparison to in vivo-derived embryos at blastocyst stage (P<0.05). In conclusion, ART manipulations may have important influences on multiple epigenetic biomarkers.