Figure 1.

Timeline for the Onset of the Neurogenic Program in hESCs

(A) Timeline and methodological details of the differentiation protocol to derive dorsal spinal sensory INs from hESCs.

(B–G) hESCs were collected for IHC and RT-qPCR analyses at day 0, 2 (B and E), 4 (C and F), and 6 (D and G) using antibodies against NANOG (red), PAX6 (green, B–D) SOX1 (green, E–G), SOX2 (blue, B–D), and DAPI (blue, E–G).

(H) hESCs rapidly exit the pluripotent state. The number of NANOG⁺ cells (p < 0.0001) and levels of NANOG transcript (O, p < 0.0001) decline by day 2 (B′) and are undetectable by day 4 (C′ and D′).

(I and J) Concomitantly hESCs enter a neurogenic state: SOX2 transcript and SOX2 protein levels remain constant (I), while SOX1 mRNA (J, p < 0.005) and SOX1 protein (J, p < 0.0001) are induced by day 2. SOX1 expression starts to decline at day 4 (J), with the number of SOX1⁺ cells decreasing at day 6. By day 6, the remaining SOX1⁺ cells are found clustered together (G). PAX6 starts to be expressed at day 4 (p < 0.01) (C‴–D‴ and K).

Two biological replicates were performed, with at least five fields of cells quantified for every IHC condition. The number of cells is expressed as a percentage of the total number of DAPI⁺ cells. Probability of similarity ^∗∗p < 0.005, ^∗∗∗p < 0.0005. Scale bar, 100 μm.