Combination of Estradiol with Leukemia Inhibitory Factor Stimulates Granulosa Cells Differentiation into Oocyte-Like Cells.

Purpose: Previous studies have documented that cumulus granulosa cells (GCs) can trans-differentiation into different non-ovarian cells, showing their multipotentiality to repopulate the injured cells in ovarian tissue. The current experiment is aimed to assess the differentiation capacity of human cumulus GCs toward the oocyte-like phenotype in vitro. Methods: GCs were isolated from healthy female volunteers subjected to in vitro fertilization or intra-cytoplasmic sperm injection (IVF-ICSI). The effect of different media supplemented with leukemia inhibitory factors (LIFs), 5 ng/mL estradiol, and 0.005 IU/mL follicle-stimulating hormone (FSH) were investigated to the differentiation of GCs toward oocyte-like phenotype via monitoring the expression of Oct3/4 and GATA-4 using flow cytometry analysis. The expression of genes such as FIGLA, NOBOX, and SYCP3 was measured by real-time polymerase chain reaction (PCR) assay. We also assess morphological adaptation by using bright-field microscopic imaging. Results: Exposure of GCs to LIFs increased the number of cells expressing stemness factor Oct3/4 coincided with the suppression of GATA-4 after 7 days (P < 0.05). We found that the transcript level of all genes FIGLA, Nobox, and SYCP-3 decreased in cells after treatment with a FSH (P < 0.05). According to our data, the incubation of GCs with estradiol increased the expression of genes related to the oocyte-like phenotype. Conclusion: Our finding revealed that the combination of LIFs and estradiol could induce the GCs' oogenesis capacity and thereby is possibly suggested as a therapeutic strategy during the occurrence of gynecological disorders.

DJ1 and microRNA-214 act synergistically to rescue myoblast cells after ischemia/reperfusion injury.

Ischemia/reperfusion injury is a tissue injury occurring post-reperfusion of tissues with pre-existing ischemia. A good blood supply to tissues aids in the survival of ischemic tissue, however, due to prolonged ischemia the levels of ATP decrease and pH declines leading to acidosis. Reduced ATP leads to an increase in the AMP/ATP ratio, causing cessation of intracellular calcium transport, hence calcium overload and cell death. In this study, we demonstrate the synergistic and antagonistic effect of DJ1 and microR-214 (miR-214) in rescuing myoblast C2C12 cells after ischemia/reperfusion in an in vitro model. Both DJ1 and miR-214 were cloned into a hypoxic inducible expression cassette and transfected into the C2C12 cells. We showed that DJ1 and miR-214 have synergistic effects in reducing intracellular lactate dehydrogenase and intracellular transient calcium levels after reoxygenation compared to control cells, in addition to reducing cell death via necrosis. Western blotting revealed a decrease in autophagosome formation in LC3II/I ratio and an increase in AKT expression in cells transfected with DJ1 and miR-214. Using quantitative real-time PCR, we demonstrated that DJ1 and miR-214 significantly reduced the expression of pro-apoptotic factors and autophagy compared to control. The results indicated DJ1 is an endogenous oxidative stress molecule and miR-214 is a potent inhibitor of the sodium calcium exchanger channel. DJ1 had the greatest effect to inhibiting mitochondrial cell death pathways by possibly acting as a modulator of autophagy. Additionally, we have concluded that miR-214 has an inhibitory effect on extrinsic cell death pathways such as necrosis and autophagy.

Distinct effect of fetal bovine serum versus follicular fluid on multipotentiality of human granulosa cells in in vitro condition.

This study aimed to develop an appropriate medium for preservation of multipotentiality in human granulosa cells. To compare the possible effect of different media supplemented with follicular fluid or fetal bovine serum, granulosa cells were cultured in vitro over a period of 14 days. Stemness feature and any alteration in the cell phenotype were monitored using colony count assay and flow cytometry analysis by monitoring the expression of Oct3/4 and GATA-4 factors. Transcript expression level of Sox-2, Klf-4, and Nanog were investigated using quantitative real-time PCR analysis. Cells were cultured in the medium supplement with follicular fluid showed normal cell morphology and epithelial-like appearance, however, cells treated with fetal bovine serum, exhibited the clonogenic potential of granulosa cells which was increased after exposure to follicular fluid after 14 days (p < 0.05). Flow cytometry analysis revealed a significant reduction in the protein level of GATA-4 in cells cultured in presence of follicular fluid compared with cells received fetal bovine serum (p < 0.001). Quantitative real-time PCR analysis disclosed reduction of Sox-2, Klf-4 and Nanog levels in cells exposed to fetal bovine serum. Our experiment showed the exposure of human granulosa cells to follicular fluid efficiently preserves the stemness characteristics of the cells.

Advantages of Sheep Infrapatellar Fat Pad Adipose Tissue Derived Stem Cells in Tissue Engineering.

PURPOSE: The goal of this study has been to evaluate adipose tissue derived stem cells (ADSCs) from infrapatellar fat pad and characterize their cell surface markers using anti-human antibodies, as adipose tissue derived stem cells (ADSCs) have great potential for cellular therapies to restore injured tissues. METHODS: Adipose tissue was obtained from infrapatellar fat pad of sheep. Surface markers evaluated by flow cytometry. In order to evaluate cell adhesion, the Polycaprolactone (PCL) was sterilized under Ultraviolet (UV) light and about 1×10(5) cells were seeded on PCL. Then, ASCs- PCL construct were evaluated by Scanning Electron Microscopy (Mira3 Te Scan, Czech Republic). RESULTS: We showed that adipose tissue derived stem cells (ADSCs) maintain their fibroblastic-like morphology during different subcultures and cell adhesion. They were positive for CD44 and CD90 markers and negative for CD31 and Cd45 markers by human antibodies. CONCLUSION: Our results suggest that ASCs surface markers can be characterized by anti-human antibodies in sheep. As stem cells, they can be used in tissue engineering.

Texosome-anchored superantigen triggers apoptosis in original ovarian cancer cells.

Texosomes, nano-endosomal vesicles, are candidates for cancer immunotherapy due to their immunostimulating properties. We designed a new structure based on texosome and staphylococcal enterotoxin B (SEB) and assessed its cytotoxic impact on an ovarian cell line. Texosomes were isolated from tumor cells, and SEB was anchored onto by protein transfer method. MTT assay and Hoechst staining were used to identify the cytotoxic and apoptotic effects of this compound on treated cells with different concentrations of texosome-SEB (TEX-SEB). Moreover, the expression rate of bcl-2, bax, bak, bcl-xl and the activity of caspase-3 and caspase-9 were investigated. Treatments of the cells with 0.5, 2.5 and 10 µg/100 µl TEX-SEB were significantly cytotoxic within 24 h (p < 0.001). Hoechst staining revealed that all tested concentrations caused apoptosis after 24 h compared with the control cells (p < 0.001). Furthermore, it was found that treatment with all examined concentrations of TEX-SEB enhanced caspase-9 activity after 24 and 48 h, while caspase-3 activity was increased upon treatment with only 0.5 and 2.5 µg/100 µl of TEX-SEB after 24 h (p < 0.001). None of the concentrations of TEX-SEB affected the expression of the cancer-promoting genes. Our construct, the TEX-SEB, is a new model being able to create cytostatic properties on cancer cells.

Exosome/staphylococcal enterotoxin B, an anti tumor compound against pancreatic cancer.

PURPOSE: Exosomes (EXOs) are acellular vehicles used for cancer immunotherapy due to their immune-inducing properties. We synthesized a novel structure based on EXOs and staphylococcal enterotoxin B (SEB) and surveyed its cytostatic effect on a pancreatic cell line. METHODS: EXOs were purified from tumor cells and SEB was anchored on it by protein transfer method. To determine the cytotoxic and apoptosis-inducing effect of this structure, treated cells with different concentrations of EXO/SEB were examined by MTT assay and Hoechst staining method. In addition, the expression rate of bcl-2, bax, bak, fas, bcl-xl and the activity of caspase-3 and caspase-9 were assessed. RESULTS: We observed that 0.5 and 2.5 µg/100µl of EXO/ SEB significantly (p<0.001) stimulated apoptosis after 24 hrs. The concentrations of 0.5 and 2.5µg/100µl of EXO/SEB raised the expression rate of bax, bak, fas (p<0.001) but had no impact on bcl-2 and bcl-xl after 48 hrs. Furthermore, it was shown that 0.5, 2.5 and 5 µg/100µl of EXO/SEB only increased the activity of caspase-3 after 48 hrs (p<0.001). CONCLUSION: Our designed structure, the EXO/SEB, is a novel model being able to induce apoptosis.

Staphylococcal entorotoxin B anchored exosome induces apoptosis in negative esterogen receptor breast cancer cells.

Exosomes (EXO) are acellular vehicles used for cancer immunotherapy due to their immune inducing properties. To identify whether designed structure based on tumoral EXO have a cytotoxic effect together with a potent immunological property, we synthesized a novel structure based on EXO and staphylococcal entrotoxin B (SEB), two immune inducer substances, and surveyed its cytostatic effect on the breast cancer cell line. EXO were purified from tumor cells and SEB was anchored on it by protein transfer method. To determine the cytotoxic and apoptosis inducing effect of this structure, treated cells with different concentrations of EXO/SEB were examined by MTT assay and Hoechst staining method. In addition, the expression rate of bcl-2, bax, bak, fas, bcl-xl and the activity of caspase-3 and caspase-9 were assessed. We observed that EXO/SEB significantly decreased the cell proliferation and stimulated apoptosis (P < 0.001) at all concentration after 24 h (P < 0.001). Furthermore, EXO/SEB raised the expression rate of bax and bak (P < 0.001) but no impact on fas and bcl-xl after 48 h. We observed reducing effect of EXO/SEB on the mRNA expression of bcl-2. After 24 h of exposing the cell with the EXO/SEB, a significant increase was found in the activity of caspase at the concentration of 2.5, 5 and 10 µg/100 µl for caspase-9 and at all concentrations for caspase-3 (P < 0.001). Our designed structure, the EXO/SEB, is a novel model for apopto-immunotherapy being able to induce apoptosis in ER(-) breast cancer cells.