Accessory Inner Segment-Like Structures Develop in Human Retinal Organoids

A novel photoreceptor structure, called the accessory inner segment (aIS), was recently identified in human rod photoreceptors. It is described as a microtubule-based, mitochondria-rich extension of the conventional inner segment attached externally to the outer segment. Human retinal organoids recapitulate many developmental and mature cellular features of the human retina. Thus, we sought to determine if retinal organoids exhibit an aIS-like structure, to support future in vitro studies of its role in photoreceptor structure and disease.

Mature retinal organoids were derived from human induced pluripotent stem cells and displayed stratified photoreceptor layers with inner- and outer segment-like processes. Immunohistochemistry and confocal imaging were used to identify aIS-like projections extending from inner segments. Electron microscopy (EM) was used to evaluate these structures in both transverse and longitudinal orientations.

Co-staining of β-tubulin with ATP synthase revealed examples of discrete microtubule- and mitochondria-rich projections, respectively, emerging apically from the inner segments of mature organoids, consistent with previously-described aIS morphology observed in cadaveric human retina. β-tubulin and GPR98 co-staining further supported the presence of aIS-associated projections, spatially distinct from the outer segment axonemes, extending from the inner segment. Transmission EM confirmed examples of aIS-like structures enriched with microtubules and mitochondria, positioned apposed to the presumptive outer segment.

Here, we provide the first evidence that aIS-like structures, previously observed only in cadaveric human retina, can be recapitulated in human retinal organoids. These findings may enable future in vitro investigations of photoreceptor structure to uncover new vulnerabilities relevant to retinal degenerations.