Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells
Transcription
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.
