Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

Take a microplate containing an adherent culture of mouse embryonic fibroblasts, or MEFs, in a medium.

The adhered MEFs function as a feeder layer, providing a supportive cell culture microenvironment.

Remove the medium and introduce human induced pluripotent stem cells, or hiPSCs, in an hiPSC medium supplemented with small molecules that enhance cell survival.

Incubate to allow hiPSCs to adhere to the feeder layer, which secretes growth factors that facilitate hiPSC proliferation.

Remove the medium and add an enzyme solution to detach the cells. Transfer the cells and centrifuge them, then remove the supernatant and resuspend them in the hiPSC medium.

Transfer the cells to a biopolymer-coated plate. Incubate to allow the MEFs to adhere.

Transfer the suspended hiPSCs to a V-bottom microplate. Centrifuge the cells and incubate them, inducing cell aggregate formation.

Replenish with a differentiation medium. The medium's nutrients and cell-cell interactions within the aggregate facilitate hiPSC differentiation into neural progenitor cells.

For this protocol, begin with establishing feeder cells. To establish this culture, plate 600,000 feeder cells into each well of a six-well plate,