Fig. 2: Specification of NSCs with ventral midbrain identity using RA and SHH signaling. | Nature Communications

Fig. 2: Specification of NSCs with ventral midbrain identity using RA and SHH signaling.

From: Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery

Fig. 2

a Schematic of hPSC differentiation with timeline of treatment with RA and SHH signaling activator (SAG) (top). All cultures were differentiated in dSMADi conditions as indicated in Fig. 1a. Immunocytochemistry for indicated markers at 9 DDC in cultures differentiated in +SAG-condition and pulsed with RA for 1-, 2-, or 4-days (bottom). b Normalized gene expression of indicated genes in 9 DDC cultures differentiated in +SAG and treated with RA for the indicated time (n = 2 independent experiments per condition). c Heatmap and hierarchical clustering of differentially expressed genes of 9 DDC cultures differentiated in +SAG and treated with RA for the indicated time (n = 2 independent experiments per condition). d Schematic of gene expression profiles defining distinct ventral progenitors in the diencephalon (Di), midbrain (MB), and hindbrain (HB). e Expression of genes associated with indicated regional progenitors at 14 DDC in cultures differentiated in +SAG + RA2D in two independent experiments. f Representative immunocytochemistry for β-CATENIN (left) and boxplots of β-CATENIN nuclear levels (right) in 2 DDC cultures differentiated in +SAG (control) and treated with RA or CHIR99021. Boxplot data: center line defines median, the box indicates quartile 1 to quartile 3, and whiskers indicate ±1.5x interquartile range. n = 227 (control), 247 (RA), 232 (CHIR99021) cells examined over 3 independent differentiations, p values derived from unpaired two-tailed t-test. g Immunocytochemistry for the indicated markers in cultures at 14 DDC differentiated in dSMADi+SAG + RA2D. h qPCR analysis of a panel of 17 genes for six independent mDA differentiations at 14 DDC (RA2D + SAG column; differentiations #1-#6). mDA samples were compared to cultures differentiated to a forebrain (RA0D + SAG, n = 2) or hindbrain (RA4D + SAG, n = 2) progenitor identity. i Quantification of the percentage of cells expressing FOXA2, LMX1A or NKX2.1 in 14 DDC cultures differentiated in +RA2D + SAG across four different hPSC lines (values, mean ± SD, n = 3–4). Scale bars: 100 µm in panels (a, g), 25 µm panel (f).

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