Graphical Description of the Experiment: Human Astrocytes during Astrogliosis (IMAGE)
Caption
(a) Derivation of human iPSC-astrocytes. Human neural stem cells (NSC) were differentiated from induced pluripotent stem cells (iPSC) and they were exposed to astrocyte induction medium (AIM). After 21 days in AIM human iPSC-derived radial glia-like cells (RCG) exhibited high proliferation rates and strong labeling for neural precursor markers. After exposing RGC to astrocyte medium for additional 4 weeks, human iPSC-derived astrocytes were obtained and used in the experiments. (b) Induction of in vitro astrogliosis. Human iPSC-derived astrocytes were exposed to serum-deprived culture medium for 24 h; then TNF-α was added to the cells in order to analyze the canonical events over the course of astrogliosis, as follows: i) NF-kB nuclear translocation was assessed 1 h after TNF-α exposure; ii) expression of cytokines was evaluated in a time window between 1.5 and 6 h following TNF-α exposure; iii) expression and secretion of cytokines and D-aspartate uptake assay were performed 24 h following TNF-α exposure; iv) densitometry of the intermediate filaments, vimentin and GFAP, cell morphology and viability, D-aspartate uptake assays were performed 5 days after TNF-α stimulation in order to investigate chronic stage of astrogliosis.
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Pablo Trindade
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