WAe009-A-1I

Figure 5. Vagal and sacral NC exhibits distinct behavior in vitro (A) Schematic drawing of experimental design. RFP-tagged hPSC line was used for vagal lineages and GFP-tagged line for sacral lineages. (B) qRT-PCR of SOX10 and HOX genes to confirm the VNC and SNC identity. n = 6 biological replicates (C) Invasion assay of VNC and SNC. Cells crossed the membrane are visualized (left) and quantified using plater reader (right). Scale bars, 50 mm. n = 3 biological replicates. (D) Migration assay of VNC and SNC on PO/LM/FN surface. Cells migrated out of spherical aggregate were imaged (left) and migration distance were quantified (right). Scale bars, 500 mm. n = 3 biological replicates. (E) 3D Matrigel embedded migration assay of co-cultured VNC and SNC. Fluorescence image of cells at 24 (upper) and 96 h (lower), show self-sorting activity. Scale bars, 500 mm. (F) Co-cultured VNC (red) and SNC (green) cells undergoing ENS differentiation with replating at day 10. Scale bars, 100 mm. (G) Heatmap of cadherins in NC samples of different axial levels. Presented as normalized counts. (H) Representative traces of electrical activity in NC-derived neurons as recorded by MEA system in over a period of 1 s.