Figure 1. An overview of the culture protocol.

The ES cells were taken out of LIF, and plated in the presence of all-trans-retinoic acid and bFGF (Step 2, denoted by 2) for 3 days followed by media without the retinoic acid but in the presence of bFGF for 14 days. These cells were then grown in suspension in the presence of N2 and B27 for 6 days (Step 3, denoted by 3) before being put into media containing DBcAMP and nicotinamide for 6 days (Step 4, denoted by 4).