Differentiating Human Embryonic Stem Cells into Oligodendrocytes
筆記録
To perform neural progenitor cell generation, culture H1 human ES cells and transdifferentiate them into neural progenitor cells, as described in the text manuscript. On day negative 1, seed 0.5 to 1 million cells in ES cell maintenance medium into each well of a 6-well plate, coated with growth factor-reduced matrix solution.
On day zero, treat cells for 24 hours with ES cell maintenance medium, supplemented with 2% DMSO. On days 1 through 6, change the full media with warm neural induction medium containing SMAD inhibitors from a commercial kit. On day 7, passage NPCs using cell detachment solution, and plate them at a seating density of 1 to 2 x 105 cells per well of a 24-well plate.
To generate oligodendrocyte precursor cells, plate the NPCs in warm neural induction medium with SMAD inhibitors from a commercial kit. On day 8, prepare a solution of 1% DMSO in the OPC differentiation medium, and treat the plated NPCs for 24 hours. On day 9, replace the media with fresh OPC differentiation medium without DMSO, then feed the cells every other day until day 15.
If the cells reach confluence before day 15, passage them to the seeding density of 1 to 2 x 105 cells per well. On day 14, plate OPCs in OPC differentiation medium at a density of 1 to 2 x 105 cells per well in a 24-well plate. On day 15, replace media with OL maturation medium.
Change the medium every other day or every day. When cells reach 90% confluence, split them at a 1-to-3 ratio for up to two passages, or until cell division slows down substantially. If OPCs divide too fast and reach confluency in less than three days, add Ara-C at a concentration of 2 to 5 micromolar for one to three days.
