Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells
DEŞİFRE METNİ
Begin by seeding clusters of hPSCs in 2 milliliters of hPSC medium containing the Rho-kinase inhibitor Y-27632 into each well of an ECM-coated six-well plate. Maintain the stem cells until they are about 50% confluent. Then, change the medium to neural medium containing SP-431542 and DMH1 to promote neural induction.
When cells are about 95% confluent, split each well 1-to-6 into new ECM-coated wells. On day 14, dissociate the cells with detachment solution, and transfer the contents of each well to a noncoated T-25 flask with medium containing Y-27632 to promote the formation of aggregates.
To generate astrocyte progenitors and astrocytes in spontaneously formed 3D aggregates, switch to neural medium containing EGF and FGF2 and feed every three to four days until astrocyte identity is confirmed at four to six months.
By default, this protocol produces dorsal cortical astrocytes. However, astrocyte subtypes can be regionally specified by the addition of patterning morphogens if desired.
Once a week, when dark centers appear, collect the spheres by centrifugation, then gently dissociate the H-astro aggregates with detachment solution. Only replate those spheres that do not spontaneously attach to avoid passaging non-CNS and non-neural cells.
