Fig. 3
Selected cultures contain V0V IN marker-expressing cells with reduced Ki67+ and other IN marker-expressing cells. A, B Representative images of day 11 Evx1-PAC mESC-derived V0V IN cultures stained by ICC for V0V IN markers Lim1 (green), Evx1 (red), and βIII tubulin (blue). Hoechst stain for nuclei is shown in montage images. C, D Representative images of day 16 Evx1-PAC mESC-derived V0V IN cultures stained by ICC for proliferative marker Ki67 (green), Hoechst for nuclei (red), and neuronal marker βIII tubulin (blue). Phase is shown in montage images. A and C show unselected cultures, while B and D show selected cultures. Scale bars are 50 µm. For each condition, magnified images (Ai, Bi, Ci, Di) are shown adjacent to their derived images to facilitate viewing details. E, F Day 11 flow cytometry analysis of selected vs unselected Evx1-PAC mESC-derived V0V IN cultures. Error bars are S.E.M., and significance is indicated as follows: * denotes p < 0.05 between selected vs unselected cultures for the same stain. E Unselected and selected samples were stained for V0V IN markers of Evx1 and Lim1 (N = 13, 15), V0C IN markers of Pitx2 and VAChT (N = 4), neuronal marker βIII tubulin (N = 11, 13), and proliferative marker Ki67 (N = 15, 10). F Unselected and selected samples were stained for markers for IN populations arising adjacent to V0V INs: Dmrt3 for dI6 INs (N = 11, 10), En1 for V1 INs (N = 7, 10), Chx10 for V2a INs (N = 10), Hb9 for MNs (N = 9, 12), and Olig2 for pMNs and oligodendroglial precursors (N = 7)