Characterization and staging of outer plexiform layer development in human retina and retinal organoids

Abstract

The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is critical for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells, and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of Bassoon to the OPL with each stage (p<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (p<0.0001). Overall, this study defines stages of human OPL development through mid-gestation and establishes HROs as a model system that recapitulates key aspects of human photoreceptor-bipolar cell synaptogenesis in vitro.

Integrating human iPSC-derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche

“…Mature RO cultures have stratified retinal layers similar to the human retina, including an outer nuclear layer (ONL) comprised solely of the nuclei of photoreceptors, an OPL comprised of synaptic junctions, and an inner nuclear layer (INL) comprised of the nuclei of second order retinal neurons and Müller glia (21) ( Fig 2b,c ). The OPL is very well conserved in the RO (22), however, the IPL and the ganglion cell layer are less well-represented in ROs because retinal ganglion cells fail to survive in great numbers to mature ages, therefore, ROs lack a pronounced layer of synaptic junctions inside of the INL (23) ( Fig 2c ). We hence refer to the layer inside of the RO INL as the lumen.…”

“…Harvesting of MPCs was as previously described (22, 23). Supernatant from the Factory containing MPCs was passed through a cell strainer (Corning, 431750).…”

The Prospects for Retinal Organoids in Treatment of Retinal Diseases

“…Cowan et al 91 compared ROs with human retina in transcriptomes, and they further characterized the functionality of ROs by measuring the light responsiveness, imaging synaptic layers, and functional synapses. Furthermore, Bharathan et al 9 applied human ROs as a model system to study the synaptogenesis in human retina, identified stages of human outer plexiform layer development, and successfully recapitulated key aspects of synaptogenesis between PRs and BCs.…”

“…Under specific culturing conditions, stem cells can be differentiated into self-assembled and layered retinal tissue spheroids that are called retinal organoids (ROs). ROs have been applied to different applications such as disease modeling,1–5 developmental biology,6–9 drug screening,10 gene therapy testing,2,11–14 and transplantation therapies 15–21. In this review, we focus on transplantation studies in recent years.…”

Prognostic Biomarkers in Uveal Melanoma: The Status Quo, Recent Advances and Future Directions

“…In the case of hiPSCs, they retain the genetic background of the donor [ 363 ]. Both hiPSCs and hESCs have allowed the study of human cells which are not normally accessible to study in the human body (for example, neurons and glial cells of the human CNS) and, therefore, have boosted the possibilities in medical research employing human cell lines, permitting the study of mechanisms of human development [ 363 , 364 ]; in vitro disease modelling, including in cancer research [ 365 , 366 , 367 ]; in the development of assays and platforms for drug screening campaigns [ 355 , 368 , 369 ]; in patient stratification and in the development of cell replacement strategies [ 355 , 363 ]. The pioneer monolayer cultures gave way to organoids, spheroids, organ-on-a-chip approaches and more recently assembloids, which employ single cell types or a multitude of different cellular types [ 355 , 360 , 370 , 371 , 372 , 373 ].…”