Fig 1: Conversion of hNES cells into hPRogFPM and their differentiation into midbrain dopaminergic neurons. a Schematic representation of the conversion and differentiation protocols. b, c RT-qPCR analysis at day 8, showing the expression of midbrain-hindbrain TFs, such as OTX2, GBX2, LMX1A, and FOXA2 (b), as well as the dopaminergic neuron markers, NR4A2, TH, SLC18A22, and SLC6A3 (c). d Immunocytochemistry analysis of the presence of OTX2 and TH in control unconverted NES cultures, compared with NES cells converted with SAF+Dkk1 and differentiated until day 8. e, f Percentage of OTX2+ and TH+ cells in the conditions in d. P = 0.02673 (e), P = 0.03233 (f), n = 3. g–i Expression of the key midbrain TFs, LMX1A, NR4A2, and PBX1, in TH+cells derived from SAI2-NES cells after conversion and differentiation. j, k TH+ cells express the mature neuronal marker, MAP2 (j), and some acquire mature neuronal morphologies, with long processes and varicosities at day 8 (k). Scale 50µm. Box plots (b, c, e, f): Center line, median; hinges, 25% and 75% quartiles; whiskers, 1.5 interquartile range. Statistics: (b, c) ANOVA, followed by pair-wise t-test with Bonferroni correction for multiple testing. (e, f). Two sample t-test; *P = 0,05; **P = 0,01; ***P = 0,001. N = 3 (GBX2, FOXA2, TH, SLC6A3), n = 4 (LMX1A, OTX2, NR4A2, SLC18A2)