Figure 2: Formation of 3D RCs.
From: Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs
(a) One NR domain (top panel) is being detached with a tungsten needle (arrowhead in bottom panel). (b) Fluorescence-activated cell sorting analysis of collected NR domains showed enrichment in NR progenitors (VSX2-positive) compared with RPE progenitors (MITF-positive). (c) Detached NR domains cultured in suspension formed 3D RCs, composed of an NR epithelium and RPE (arrow). (d) Higher magnification of a typical 3D RC with an NR epithelium continuous with the adjacent RPE bundled at the tip (arrowheads). (e,f) The pseudostratified neural epithelium within the RC showed the typical polarity, with mitosis (Phospho-Histone H3 (PH3)-positive) occurring at the apical side and postmitotic neuronal precursors (HU C/D-positive) accumulating at the basal side. (g–i) NR cells proliferated actively (EdU-positive, g) and co-expressed transcription factors characteristic of neural retina progenitor cells (h,i). (j–k) Retinal progenitors within the NR epithelium underwent interkinetic nuclear migration. (j) Time-lapse imaging of retinal progenitors expressing nuclear green fluorescent protein (GFP). (k) Three-dimensional volume rendering of the cells shown in (j); red dot: cell undergoing mitosis; yellow dot: cell nucleus migrating from the apical to the basal side of the neuroepithelium; blue dots: cells undergoing apoptosis. Scale bars, 100 μm (a and c); 50 μm (d–i).
