Figure 2.
Induction of FBs into iENPs by Six TFs Selected Using the PAX6:EGFP Neural Reporter
(A) Schematic depiction of the experimental strategy for reducing the number of TFs (from 25 to 6) for iENP induction using the neural reporter PAX6:EGFP.
(B and C) Stepwise selection of potent iENP factors for iENP-6F generation by single TF dropouts from the original 25-TF set (B) and the 15-TF set (C). The results are expressed as the relative percentage of PAX6:EGFP+ cells after each TF was removed from the 25-TF or 15-TF combinations.
(D) Comparison of the efficiency of induction of PAX6:EGFP+ cells from FBs by 25-, 15-, and 6-TF combinations.
(E) Global gene expression heatmap of FB, hESC-ENP, iENP-6F, and iENP-15F as determined by microarray analysis.
(F) ICC staining of iENPs-6F using antibodies against the indicated NP markers.
(G) RT-PCR analysis of endogenous and exogenous expression of the 6 TFs using mRNA isolated from iENP-6F.
(H) RT-PCR analysis of the indicated neural genes using mRNA isolated from iENP-6F.
FB, fibroblast; NC, negative control (H2O); plasmid, expression plasmids for the indicated genes. All quantitative data were obtained from three independent experiments and are presented as means ± SD. ∗p < 0.05. See also Figures S2–S4.