FIG. 3.

A stage-related comparison of gene expression between the experiments with (+) and (−) outcome regarding the Th expression revealed milestones with predictive potential. (A) mRNA expression levels of neural progenitor markers, Pax6, Sox1, Nestin, and Ncad, VM markers, En1, Otx2, Foxa2, and Lmx1a as well as pan-neuronal markers, Tau and Dcx, in negative (−) experiments, relative to positive (+) experiments. Mean±SEM; n=3. *P<0.05 (Student t-test). (B) Immunostaining for Nestin in stage II, Tau and Nestin in stage III, Otx2 in stage IV, and Th in stage V in 2 representative (+) and (−) experiments. (C) Relative mRNA levels in stage V of the (+) experiments as compared to the (−) ones for the dopaminergic (Vmat2, Girk2, and Calbindin), GABAergic (Gad1), glutamatergic (vGlut2), cholinergic (Vacht), and glial (Gfap and Osp) markers. Mean±SEM; n=3. *P<0.05, **P<0.01 (Student t-test). (D) Immunostaining for Tau, Th, Gfap, Osp, GABA, Glutamate, and Vacht confirmed their expression in stage V in one representative (+) experiment. (E) Schematic representation of 5-stage protocol showing the gene expression levels in each stage that predict positive (+, green) and negative (−, red) end point outcome regarding the Th expression. The milestones are represented as mean ΔCt values (related to GAPDH). When a milestone is not reached, the prediction for a (−) outcome is followed by stopping the experiment (STOP). (B, D) Nuclear staining with DAPI (blue); scale bar: 20 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′, 6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/scd