FIG. 5.
Patterning floor plate-derived midbrain dopaminergic neurons. (A) Time line illustrating the protocol for basic NP dual-SMAD differentiation and DAergic patterning (DAergic). Expression of Cj-ESC (B) and Cj-iPSC (C) markers after 12 or 16 days of patterning. The nonpatterned controls default to a dorsal forebrain phenotype judged by PAX6+/FOXA2− (B1, C1) and OTX2+/EN1− (B4, C4). Sufficient ventralization was observed after 16 days of patterning compared to 12 days in both cell lines (B2,3, C2,3). Posteriorization to a midbrain fate was sufficient and maintained after 12 and 16 days of patterning, respectively, in both lines (B5,6, C5,6). The Cj-ESC and Cj-iPSC lines only produced TH+ neurons after the DAergic protocol (B7,8, C7,8). Replacing SHH with the small-molecule purmorphamine from day 4 to 28 (E) led to a visible increase in FOXA2 expression by day 8 and 12 with maintenance after day 27, with no change in posteriorization patterning (F–J). FOXA2 was maintained after 49 days and observed in TH+ neuronal nuclei (K). Scale bars: B1, C4–6, 50 μm; B2–8, C1–3,7,8 (E–K), 100 μm.