Enhancing Stem Cell Differentiation with a Transient DMSO Treatment

Pretreat cells with DMSO as described for 2D cultures and prepare Noggin and SP431542 stock solutions. Prepare enough 10% knockout serum replacement or KOSR in knockout DMEM for three to four days of media change. Prepare ectodermal differentiation media by adding Noggin and SP431542 to prewarmed KOSR knockout DMEM as described in the manuscript.

After DMSO pretreatment, aspirate media from cells, and add 2 milliliters of differentiation media to each well. Allow the cells to incubate for three to four days at 37 degrees Celsius in a carbon dioxide incubator, replacing media daily with fresh differentiation factors.