This article describes a protocol for differentiating mouse embryonic stem cell-derived neural progenitor cells into neurons. The process involves the use of a gelatin-coated multiwell plate and a nutrient-rich basal differentiation medium supplemented with neural growth factors.
Begin with a gelatin-coated multiwell plate containing mouse embryonic stem cell-derived neural progenitor cells in a nutrient-rich basal differentiation medium.
The progenitor cells utilize these nutrients from the media to maintain their viability.
Incubate to allow cell attachment to the gelatin-coated surface, then wash to remove unattached cells and debris.
Introduce a basal differentiation medium supplemented with a stabilizing agent, additional nutrients, and neural growth factors crucial for cell differentiation and then incubate.
The progenitor cells consume the nutrients and growth factors, initiating their differentiation into neurons that begin to extend their cellular processes.
Concurrently, the stabilizing agent interacts with toxic metabolites and protects the developing neurons from oxidative damage, preserving their viability.
Regularly replace the media to maintain nutrient levels.
Over time, the neurons mature, developing axons — the longer cellular processes.
Additionally, they also develop dendrites, the shorter cellular processes, confirming stem cell differentiation into neurons.
Seed about 500,000 mouse embryonic stem cell derivatives in 2 milliliters of basal differentiation medium per well onto the 0.1% gelatin-coated plates. Randomly divide the derivatives into three groups in order to test three phase II differentiation protocols. Incubate the plate for six hours. Then, wash the cells twice with 2 milliliters of PBS and add 2 milliliters of basal differentiation medium, N2 B27 medium 1, or N2 B27 medium 2 to each well, depending on the protocol. Place the plates into the incubator, and allow the cells to differentiate for another 10 days, changing the corresponding medium every two days.
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