Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells

Begin with a low-attachment well plate containing human-induced pluripotent stem cells or iPSCs in a stem cell medium supplemented with a Rho-kinase inhibitor.

The inhibitors enter the cells, block the Rho-kinase enzymes, and maintain cell viability. This promotes the formation of iPSC aggregates.

Replace the medium with a stem cell medium to allow aggregates to grow. Transfer them into a low-attachment culture dish with a cortical induction medium.

The components of this medium cause the iPSCs to differentiate into neuroectodermal cells, a neural precursor.

Transfer each neuroectodermal aggregate onto a film with shallow wells.

Replace the medium with a basement membrane matrix and incubate to allow matrix solidification and entrapment of aggregates.

Transfer these matrix-embedded aggregates to a dish containing a cortical induction medium and incubate with agitation.

Over time, the neuroectodermal cells utilize the differentiation factors and develop into neuroepithelial cells.

Change the medium to an organoid differentiation medium, which allows the cells to mature into more specialized cell types, forming a cerebral organoid that resembles the forebrain cortex.