Fig. 1

Overview schematic of basal cholinergic differentiation protocol. a Cells are plated and allowed to reach 100% confluency (Day 0), before the initiation of dual smad inhibition and the subsequent introduction of ventralizing agents (Day 2). At day 10 the monolayer is dissociated, sorted for p75+ cells, and kept as NEBs until day 19. Then the culture is dissociated again into a monolayer (See Methods for more details). b Left panel shows sustained EGFP expression driven by Nkx2.1 induction in NKx2.1-EGFP hESCs upon SHH plus purmorphamine or SAG plus purmorphamine treatment, maintained at Day 14, after removal of treatment at Day 8. Right panel shows Nkx2.1, Lhx8 and BF1 relative gene expression to GAPDH measured by qPCR, in NKx2.1-EGFP cell line in the presence of the indicated ventralizing agents, or unpatterned (UNP) at Day 12. n = 3, in technical triplicates. c Confocal microscope images of Nestin (green), Sox2(red) and DRAQ5 (blue) immunostaining in fControl and control lines at Day 11, showing typical neural rosettes (left panel), or Tuj1 (green), Nkx2.1 (red)right and DRAQ5(blue) in the right panel. Images representative of 3 independent experiments. d Fluorescence microscope images of immunostained NEB cryosections or dissociated NEBs into a monolayer with the BFCN markers Nkx2.1/Tuj1/p75/BF1/MAP2/ChAT. e Dissociated NEBs into a monolayer immunostained at Day 50 with MAP2(green), ChAT(red) and Hoescht (blue). Fluorescence microscope images the effect of NGF addition to SAG plus purmorphamine treatment alone. Images are representative of at least 3 independent experiments