Project description:Exosomes endogenous microRNAs (miRNAs) play critical roles in many biological processes.to obtain profiles of the miRNAs of exosomal and cell lysates in ovarian cancer cell lines.
Project description:We collected ovarian follicle fluids from 68 patients and assigned them to good group or bad group according to their oocyte quality. The exosomes were isolated and characterized. Exosomal microRNAs were extracted, the library was constructed and sequenced by Illumina hiseq platform. The exosomal microRNA expression was analyzed and profiled, the target genes were predicted, GO terms were enriched by GOSeq and KEGG pathway was analyzed using miranda.A total of 47 differential microRNAs was expressed significantly between good and bad group, of which 9 microRNAs were known microRNAs and 7 of them was upregulated in the bad group. In-silico analysis indicated that several of these exosomal microRNAs were involved in pathways implicated in oocyte quality.Our study suggests that exosomal microRNAs in ovarian follicle fluid are critical in maintaining the oocyte quality. Our study greatly improve our understanding of exosomal microRNAs in human ovarian follicular fluid, paving the way for further investigation on the microRNA functions in the ovarian microenvironment and the mechanism behind it.
Project description:To check the profile of exosomal and cellular miRNA in ovarian cancer cell lines, total RNA were extracted from exosomes and cells. Thirteen ovarian cancer cell lines (A2780, ES-2, CAOV3, SKOV3, OV-90, OAW42, MCAS, COV362, RMG-1, RMUG-S, KURAMOCHI, NIH-OVCAR3 and A2780cis) were investigated, and HOSE1, HOSE2 and HOSE3 (human ovarian surface epithelim cell lines) were used as control.
Project description:Spent culture media was collected on day 5 after in vitro fertilization from 5 human blastocysts that were subsequently transferred or frozen. Corresponding blank culture media from the same lot were cultured alongside the embryos and used as negative controls along with PBS. The samples were prepared for total RNA sequencing using SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) and for small RNA sequencing using the QIAseq RNA Library Prep kit (Qiagen). Prepared libraries were sequenced as 100 bp paired end on an Illumina HiSeq 4000 sequencer for total RNA and Illumina NextSeq500 sequencer for small RNA. We found that total RNA mapped only 3% of the reads to gene regions in spent culture media and control and only 2% in PBS samples mapped to gene regions. For the small RNA analysis, human annotated miRNAs constituted from 0.1% to 1.4% of the mapped reads and 40% of small RNA reads mapped to the human genome. tiRNAs derived from 5 different mature tiRNA were differentially expressed when comparing conditioned to unconditioned media. We conclude, that in spite of applying state-of-the-art sensitive detection methods no miRNAs were found to be reliably present in the spent culture medium. In contrast, tiRNA fragments appear to be overexpressed in cultured IVF media samples.
Project description:In the field of in vitro embryo production it is generally thought that the culture media are not as good as the oviductal fluid, resulting in detrimental changes of embryonic gene expression. Yet in vitro embryo culture (IVC) is one of the pillars of the assisted reproductive technologies. Therefore, unintended effects on gene expression may impact on the health of ART babies (PMID 27554442). Granted, human ART has many more components than just the culture media, but, the practice of embryo culture superimposes with the phase of life when cellular totipotency is present - that's why embryo culture media receive special attention. Discerning what culture media do is difficult because several confounders are present, including but not limited to oocyte heterogeneity, sperm heterogeneity as well as time of fertilization. We propose to take advantage of experimentally produced parthenogenetic embryos, and thereby remove two confounders (sperm variability, time of fertilization), in order to assess the effects of specific culture media of embryonic gene expression, in the mouse model of human reproduction. After synchronous parthenogenetic activation of MII oocytes, pronuclear oocytes were cultured in parallel in ART medium vs KSOM(aa) medium. Resultant blastocysts were compared and contrasted for gene expression among the parthenotes, as well as against zygotic blastocysts.
Project description:Abstract The therapeutic properties of extracellular vesicles (EVs) derived from stem cells and stem-like cells make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies heavily on the ability to manufacture EVs in a scalable, reproducible, cGMP-compliant manner. The choice of cell culture media is a critical component for EV manufacturing. In this study, we used human amniotic epithelial cells (hAECs) as a cell model system to explore the effect of chemically defined serum-free media on EV production. Here we showed that different culture media and different cell culture supplements have variable effects on EV production including culture parameters, EV yield, and EV biogenesis. Cell viability and proliferation rate are not reliable quality indicators of EV manufacturing. The levels of common EV tetraspanins and epitope makers can be impacted by different culture media formulations even when culture parameters are maintained, and EV yield are comparable. This study has uncovered some critical aspects regarding EV production culture media that need to be considered in clinical-grade scalable EV manufacturing.
Project description:Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimics the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provides more realistic levels of lipids. Here we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. Mechanism of action studies indicate that the ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets DEGS1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies reveal ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and suggest a general screening strategy for identifying additional cancer dependencies masked by culture conditions unrepresentative of the in vivo environment.