A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Take mouse embryonic stem cells, or mESCs, suspended in a differentiation medium, and incubate.

In the suspension culture, mESCs aggregate to form a structure termed embryoid bodies or EBs.

EBs are three-dimensional aggregates that mimic a developing embryo and can be differentiated into neural cells.

Transfer the EBs into a tube and allow them to settle by gravity.

Remove the nutrient-depleted medium; resuspend the EBs in a fresh medium, and transfer them to a culture dish.

Incubate the suspension culture to facilitate the growth of EBs.

Transfer the EBs suspended in the medium onto a multiwell plate. The wells are coated with an adhesion protein, allowing the EBs to attach.

Introduce retinoic acid, which enters the cells and triggers the formation of a transcription complex.

The complex induces gene expression, promoting the differentiation of the cells into neural progenitor cells, or NPCs, which exhibit extended cellular processes.

Add 1.5 million mouse embryonic stem cells into a non-adhesive bacterial dish in 10 milliliters of basal differentiation medium, and incubate the plate at 37 degrees Celsius and 5% carbon dioxide to allow for embryoid body formation.

After two days, transfer the cell aggregates into 15-milliliter centrifuge tubes, and let them settle by gravity. Remove the supernatant and add 10 milliliters of fresh basal differentiation medium to resuspend the embryoid bodies. Then, replant them into a new non-adhesive dish and incubate them for another two days.

Collect and seed about 50 embryoid bodies in 2 milliliters of basal differentiation medium per well onto a gelatin-coated six-well plate. For retinoic acid induction, add 2 microliters of RHA stock into each well to a final concentration of 1 micromolar. Return the plate to the incubator and differentiate the cells for another four days.