This article outlines a protocol for differentiating human pluripotent stem cells into astrocyte progenitors and astrocytes using Rho-kinase inhibitors and a neural differentiation medium. The process involves seeding stem cells, inducing differentiation, and maintaining cell viability through specific treatments.
Begin with an ECM-coated plate and seed clusters of human pluripotent stem cells in a growth medium containing Rho-kinase inhibitors.
ECM enhances cell attachment, while the inhibitors block Rho-kinases, preventing apoptotic cell death and promoting cell survival and proliferation.
Introduce a neural differentiation medium to induce stem cell differentiation into neural progenitor cells.
Treat with a detachment solution; the enzymes in this solution degrade the ECM and cell contact, releasing the cells.
Transfer them into a non-coated flask containing Rho-kinase inhibitors.
The inhibitors facilitate cell survival and spontaneous aggregation into three-dimensional structures or spheroids.
Supplement the spheroids with a growth factor-rich neural differentiation medium, allowing neural progenitor cells to differentiate into astrocyte progenitors and form astrospheres.
Regularly, add a detachment solution to dissociate the astrospheres into smaller clusters. This allows nutrients to reach the inner cells of the spheroid and maintain their viability.
Maintain the astrospheres in a fresh medium for progenitors to mature into astrocytes.
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.
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