Figure 1. Directed differentiation of human ES and iPS cells to cortical stem and progenitor cells.

A. Over the 15 day neural induction period, Oct4-expressing hES cells differentiate at high efficiency to Pax6-expressing neural stem cells. Asterisks indicate the absence of detectable Pax6-expressing cells at day 0 and of Oct4-expressing cells at day 15. Error bar, s.e.m., n=3 samples for each marker at each timepoint.

B. Quantitative RT-PCR for the cortical stem cell-expressed transcription factor Foxg1 demonstrates that the induction of cortical stem cells begins after 5 days and peaks after 20 days, whereas Tbr2-expressing intermediate progenitor cells and newly-born neurons begin to appear almost a week later. Error bars, s.e.m. Semi-quantitative RT-PCR for the cortical stem cell-expressed Emx1 and the ventral and/or caudally expressed transcription factors Dlx1, Nkx2.1, HoxB4 and Isl1 further shows that neural rosettes generated by this method do not express mRNAs for the ventrally and caudally expressed genes. This is in contrast with rosettes ventralised by treatment with the hedgehog agonist purmorphamine (hiPSCs + Pur.).

C. Confirmation of the cortical identity of hESC-derived stem and progenitor cells (Ki67-positive) by expression of proteins characteristic of cortical stem cells: Pax6, the intermediate filament protein Vimentin and Otx1/2. Scale bars, 50 μm.

D. Quantification of the efficiency of cortical induction, as assayed by the percentage of Pax6-expressing cells (percentage of nuclei, detected with DAPI), in the presence or absence of retinoids in two hESC and four hiPSC cell lines. Values are the average of three cultures for each cell line. Error bars, s.e.m.

E-P Phase contrast and Pax6 immunofluorescence images of hESC and iPSC-derived cultures 12-14 days after the initiation of neural differentiation by dual SMAD inhibition in the absence or presence of exogenous retinoids. Neural rosettes (arrows) were infrequently observed when retinoids were not added to the cultures. Small clusters of Pax6-expressing cells were also observed in these cultures. Efficient induction of neuroepithelial rosettes was observed in all lines, with the majority of cells expressing Pax6. Scale bars, 50 μm.