Figure 5

Formation of laminated retinal organoids with OS-like structures and expression of proteins associated with mature PRs. (A) Immunohistochemical staining of retinal organoids for ARR3, ARL13B, PRPH2 show morphological maturation and apical organisation of developing photoreceptor segment-like structures. Retinal organoids generated in AMMs using animal-derived supplements (xeno) or xeno-free (XF) conditions, transferred to suspension culture from day 60 of differentiation or maintained in AMMs showed immunostaining for cone outer segment protein ARR3. Retinal organoids matured in suspension in xeno conditions developed prominent OS-like structures labelling with ARR3, ARL13B or PRPH2. (B) Bright-field image of whole mount retinal organoid at week 32 of differentiation generated from MShef10 ESC line using AMMs and animal-derived supplements (xeno) and cultured in suspension; OS-like structures (arrowhead) developed from week 22–25 of differentiation. (C) IS, nascent OS, OLM, bb, cc and mt were detected in TEM images taken from ultrathin sections of week 32 retinal organoids (cultured in AMMs and grown in suspension under xeno conditions; n = 2) and of week 40 retinal organoids (AMMs in suspension under xeno-free conditions; n = 2) (MShef4 ESC line); cc, connecting cilia; OS, outer segment; IS, inner segment; bb, basal body; mt, mitochondria, OLM, outer limiting membrane, tj, tight junction.