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Exosomes are extracellular vesicles ranging in size from 40 to 100nm that are used for extracellular communication to control the cell niche environment with regards to differentiation. Initial studies in our lab have shown that exosomes isolated from differentiating neurites can cause differentiation in cells absent typical neuronal hormones. However, these studies only used exosomes isolated after complete differentiation. To further clarify the role of exosomes in cell differentiation, we isolated exosomes from NGF-treated PC12 cells after two, four and six days. The exosomes were taken at the specific times due to a slight majority of cells showing differentiation at day three, with an overwhelming majority showing differentiation at day six. Day two, four and six were thus good markers in the development of cell differentiation. Each set of exosomes was added to a different well of untreated cells, which were then observed for differentiation daily over a six-day period. The results showed a considerably larger amount of percent differentiation in cells treated with the day six exosomes compared to cells treated by the day two and day four exosomes. The change in number of differentiated cells in different exosome treatments indicate that exosomal contents change over time. In an effort to make sure that no NGF contamination occurred in our treatments, an NGF-ELISA was completed testing all the treatment exosomes with results showing no NGF. Additionally, exosomes were isolated from NGF spiked media to show that no NGF was carried over in our exosome isolation process. Both tests further evidence that our exosomes did not contain NGF. Our overall results indicate that the isolated exosomes changed contents over time and that their contents caused greater percent differentiation over time. Funding provided by the Cell Biology Education Consortium (CBEC) and through the AR-EPSCoR Center for Advanced Surface Engineering.