Methods for differentiating dopaminergic neurons from stem cells

A method of differentiating pluripotent stem cells into dopaminergic neuronal progenitor cells, the method comprising:

• a) performing a first incubation comprising non-adherently culturing pluripotent stem cells in a first multiwell culture vessel under conditions to produce a cellular spheroid, wherein the first incubation comprises:
• (i) exposing the pluripotent stem cells to at least one inhibitor of TGF-β/activin-Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for at least one day, wherein Day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling, in the absence of:
• x) an activator of Sonic Hedgehog (SHH) signaling, and y) an inhibitor of glycogen synthase kinase 3ß (GSK3ß) signaling;
• and
• (ii) starting on Day 1 of the first incubation, wherein Day 1 is one day after the first day that the pluripotent stem cells are first exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling, exposing the cells obtained from step (i) to at least one activator of Sonic Hedgehog (SHH) signaling and at least one inhibitor of glycogen synthase kinase 3β (GSK3β) to produce the cellular spheroid;
• and
• b) performing a second incubation comprising adherently culturing cells of the spheroid in a second culture vessel under conditions to further differentiate the cells into dopaminergic neuronal progenitor cells;
• wherein the dopaminergic neuronal progenitor cells exhibit increased expression of FOXA2 and CORIN and decreased expression of NKX2.1 and PITX2 compared to cells differentiated in a method that comprises performing the first incubation in adherent culture.