Although, the co-expression of TH and TUJ1 defines the stable phenotype of the produced DA neurons, studies have proven an additional role of the forkhead transcription factor, FoxA2 in maintaining this stability (Ferri et al., 2007; Kittappa et al., 2007). Further modifications were implemented using new phenotypic markers to show that ventral midbrain DA neurons were not previously obtained, and thereby three modifications were added to the previously established differentiation protocol (Sonntag et al., 2007) in order to generate DA neurons with maintained stable phenotype (Cooper et al., 2010). First, retinoic acid (RA) was added at an early phase and at low dose to improve the regional identity of neural progenitor cells. Second, a high activity form of human SHH was used to permit production of a large population of FOXA2+ neural progenitor cells in vitro. Finally, FGF8b was replaced by FGF8a, and WNT1 was added for robust generation of FOXA2+ DA neurons (Cooper et al., 2010).
