Figure 1.
FOXA2 expression in midbrain dopamine neurons derived from human pluripotent cells. (A): DA differentiation paradigm for neural induction of human PSCs lines (human embryonic stem cells: H9, ES3, ES5, and iPSC C1; see supplemental online Fig. 1). (B): Transcriptional profile by quantitative real-time polymerase chain reaction shows early induction of FOXA2 and the region-specific transcription factors En1 and OTX2. (C–E): Expression of FOXA2 in neural progenitors and neurons was corroborated by immunofluorescence in all PSCs lines by coexpression with Nestin, TuJ1, and TH at corresponding differentiation stages. (F): βIII tubulin and TH protein content increased in the cultures upon differentiation, shown in a representative Western Blot. (G): Quantification of the percentage of THpos cells in cultures at early (4 weeks) and late (>6 weeks) differentiation stages. (H): THpos cells also expressed other ventral markers like OTX2 (see also supplemental online Fig. 1) Scale bars = 100 μm. Abbreviations: AA, ascorbic acid; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CHIR, 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; DAPI, 4[prime],6-diamidino-2-phenylindole; diff, differentiation; div or DIV, day in vitro; FBN, fibronectin; FGF, fibroblast growth factor; GDNF, glial cell-derived neurotrophic factor; GEL, gelatin; iPSC, induced pluripotent stem cell; LDN, 4-[6-(4-Piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline hydrochloride; POL, polyornithine; SB, 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide hydrate; SAG, N-methyl-N′-(3-pyridinylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane; TGF, transforming growth factor; TH, tyrosine hydroxylase; tub, tubulin.