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. 2017 Sep 8;6:e24502. doi: 10.7554/eLife.24502

Figure 2. Inhibition of neural specification of hESC by ID-8.

(A) design of time course experiments. Dual SMAD inhibitor induction was carried out from Day 0–7 and cells were assayed for PAX6 expression on Day 16; ID-8 was added from Day 0–16, Day 0–7, Day 0–9, or Day 9–16. (B) phase contrast micrographs showing morphology of control hESC, neural progenitors induced by dual SMAD inhibition, and neural progenitors incubated with 0.5 µM ID-8 throughout the neural induction protocol, on Day 16. (C) dose response study of the inhibition of induction of PAX 6 positive cells by ID-8 showing flow cytometry profile (left panel) and percentage of PAX6 positive cells (right panel). Error bars, SD; *p<0.05, **p<0.0.01. (D) effect of timing of ID-8 exposure at 0.5 µM on inhibition of PAX6 induction. Error bars, SD; **p<0.05, *p<0.0.01. (E) indirect immunofluorescence analysis of NESTIN (green) and POU5F1 expression (red) in control hESC, neural progenitors, and cultures subjected to neural induction in the presence of 5.0 µM ID-8. Nuclear counterstain, dark blue. (F) flow cytometry profiles showing expression of stem cell surface molecules GCMT-2 and CD9 in control cells, neural progenitors and cultures subjected to neural induction in the presence of 5.0 µM ID-8. A-D, studies carried out with HES3 (PAX6mCherry) cell line; E-F, WA09 hESC.

Figure 2.

Figure 2—figure supplement 1. ID-8 treatment does not interfere with hESC proliferation or alter expression of stem cell markers under conditions that promote self-renewal.

Figure 2—figure supplement 1.

(A) flow cytometry profiles of control and ID-8 treated cells following incubation with a pulse of Edu. (B) proportion of cells in GO/G1, S, and G2/M phases in control or ID-8 treated cultures; (C) ID-8 treatment does not alter proportion of cells bearing hESC stem cell surface markers. Dual label flow cytometry profiles for stem cell antigens GCTM-2 and CD9 in control and. ID-8 treated cells.
Figure 2—figure supplement 2. Targeting of the PAX6 gene with an mCherry-Ires-Puro reporter cassette.

Figure 2—figure supplement 2.

(A) Structure of the PAX6 targeting vector. The upper line shows the endogenous PAX6 gene with exons (and exon number, e12, e13, e14) indicated by grey boxes. The second line shows the targeting vector with its constituent components, from left to right, the 5’ homology arm, a T2A sequence (2), mCherry (Ch), IRES (I), puromycin resistance gene (P), a loxP flanked PGKNeo cassette (Neo) and the 3’ homology arm. The third line shows the structure of the modified PAX6 locus following integration of the targeting vector. The forth line shows the modified PAX6 locus following cre-recombinase mediated removal of the PGKNeo selection cassette. The protein products predicted to be translated from this locus, PAX6, mCherry and Puromycin resistance, are shown underneath. (B) PCR analysis confirming correct integration of the targeting vector in two independent clones (85, 163). Note these PCR products of the correct size were not generated when wild type (wt) was used as a template.