Chunk #46 — ONLINE METHODS — Astrocyte Differentiation of hiPSCs and Treatment of Neurons from ApoE-deficient hiPSCs with Conditioned Media from Astrocyte Culture
hiPSCs were differentiated into astrocyte progenitors and astrocytes as previously reported with some modifications59. The astrocyte progenitors were generated using the procedure as described for neuronal differentiation of hiPSCs. Instead, these progenitor spheres were kept in suspension for further expansion in the presence of 10 ng/ml bFGF (PeproTech) and 10 ng/mL EGF (PeproTech), and passaged by disaggregating into small clusters with a Pasteur pipette for around 90 days. For astroglial differentiation, progenitor spheres were dissociated with accutase to single cells, and seeded onto the plates coated with PLL and laminin in Neural Differentiation Medium without bFGF and EGF. The astrocyte conditioned media (ACM) were collected from the astrocyte culture, and the apoE levels of these media were measured with Human ApoE (AD2) ELISA kit (Cat # EHAPOE, Thermo Scientific). The amount of ACM for treatment of the neurons derived from apoE-deficient hiPSCs were adjusted to the same apoE concentration (0.35 nM or 1.47 nM), based on apoE levels in the ACM collected from apoE3/3 or apoE4/4 astrocyte culture. After ACM treatment for 1 week, the media and cell lysates of neurons derived from apoE-deficient hiPSCs were collected and further analyzed.
