Figure 3. Cortical rosettes differentiated from PSCs generate basal progenitor and outer radial glial cells.

A-C. The majority of cells within hESC (A) and hiPSC (B, C)-derived cortical rosettes are Vimentin-expressing neuroepithelial cells with apical processes (arrows) oriented to the centre of each rosette (asterisks). Scale bar, 50 μm.

D-F. A subpopulation of rosette cells towards the periphery of each rosette express the basal progenitor cell/subventricular zone cell transcription factor Tbr2. A subset of Tbr2+ cells are proliferating cells, as they co-express Ki67 (white arrowheads), whereas others do not (yellow arrows). Scale bar, 50 μm.

G-I. Many of the Tbr2+ cells are newly-born neurons, as they also express Doublecortin (yellow arrows). Scale bar, 50 μm.

J-L. Whereas the majority of mitoses (phospho-histone H3+ cells) are located apically, at the centre of each rosette (yellow arrows), mitoses are also found displaced from the centre or towards the periphery of rosettes (white arrows). These abventricular mitoses indicate the presence of a secondary, basal progenitor cell population within the rosettes. Scale bar, 50 μm.

M, N. Quantification of the proportions of Tbr2+ cells found in cortical rosettes – between 15 and 20% of cells within rosettes derived from different hESC and hiPSC lines express Tbr2 (M). Of the Tbr2+ population, approximately 40% are Ki67+ cycling progenitor cells (N). Error bars, SD.

O. Quantification of the relative proportion of abventricular, basal cortical stem/progenitor cell mitoses (phospho-histone H3+ cells) occurring in cortical rosettes generated from hESCs (H9) and two hiPSC lines. Phospho-histone H3+ mitotic cells were defined as abventricular if found more than 5 cell diameters from the luminal surface. Error bars, SD.

P-R. Representative images of GFP-labelled individual radial glial (ventricular) cells (P) and oRG cells (Q, R). The radial glial cell (P) expresses Pax6 protein (blue nucleus) has an apical process (red arrowhead, P) that contains a centrosome at its apical extreme (yellow arrow) and a long basal process (white arrowhead, P). In contrast, oRG cells (Q, R), which also express Pax6 (blue nuclei), have a single basal process (white arrowheads) and their centrosomes are found in the cell body, near the nucleus (yellow arrows). Scale bars: P, 25 μm; Q, R, 10 μm.

S-U. Two types of mitotic progenitor cells are found in abventricular rosette locations: Pax6-expressing cells with phospho-vimentin+ basal processes (white arrowheads, S, T) that do not express Tbr2 (white arrows, U), which correspond to oRG cells; and Pax6-negative cells lacking basal processes (yellow arrows, T) that express Tbr2 (yellow arrowhead, T) and correspond to basal progenitor cells. Scale bars: S, T, 20 μm; U, 25 μm.