This article describes a method for differentiating human pluripotent stem cells into ectodermal precursors with neural differentiation ability. The process involves the use of DMSO to arrest cells in the G1 phase of the cell cycle and the application of specific signaling pathway inhibitors.
Begin with a multi-well plate containing adhered human pluripotent stem cells with multilineage differentiation ability.
Introduce a stem cell medium containing dimethyl sulfoxide or DMSO.
DMSO enters the cells and prevents the progression of the cell cycle from the Gap 1, or G1 phase, an initial stage of the cell cycle, to the synthesis, or S phase.
This creates a uniform population of cells in the G1 phase, enhancing their differentiation ability.
Replace the medium with a nutrient-rich ectodermal differentiation medium containing inhibitors of the BMP and TGF-β signaling pathways.
These inhibitors bind to specific ALK receptors on the stem cells, blocking the BMP and TGF-β signaling pathways and preventing their differentiation into endoderm and mesoderm precursors.
This allows the selective differentiation of G1-arrested stem cells into ectodermal precursors possessing neural differentiation ability.
Regularly replace the medium with a fresh medium to maintain the nutrient and differentiation factor levels, ensuring cell survival.
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.
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