![]() ![]() Spatial distribution of presynaptic ribbon proteins by OPL stage in human fetal retina. Asterisks indicate nascent layers in OPL-Stage 1. ![]() Arrowheads: developing OPL (E,I,M,Q) HC processes and DBC dendrites (F,J,N,R). Arrows: spatial location of nascent HCs and DBCs (C) a single row of HCs (Q) maturing cones beginning to express ARR3 (P). Stage-specific features are pointed out with yellow arrows and arrowheads. RXRγ expression is also detected in RGCs in the GCL (D,G,K,O,S). Of note, a subset of RGCs in the GCL (F,N,R), amacrine cells in the INL (R) and Müller glia in the INL (R) expressed CALB. Cell-type specific markers were used in combination to identify photoreceptor precursors (RCVRN +VSX2 − in B RXRγ + and NRL + in D,G,K,O,S), non-photoreceptor cells (RCVRN −VSX2 + in B), HCs (CALB + in C,F,J,N,R), DBCs (PCP2 + in C,F,J,N,R), maturing cones (ARR3 + in H,L,P,T) and maturing rods (RHO + in H,L,P,T). (A-T) Representative immunofluorescence images of a fetal week 16.6 (∼D118) retina showing cellular morphogenesis at OPL-Stage 0 (A-D), OPL-Stage 1 (E-H), OPL-Stage 2 (I-L), OPL-Stage 3 (M-P) and OPL-Stage 4 (Q-T). Staging of OPL development in human fetal retina. Published by The Company of Biologists Ltd. 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.īipolar cell Human fetal retina Human retinal organoid Outer plexiform layer Photoreceptor Ribbon synapse. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (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. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P<0.0001). 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. 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 crucial for visual processing but poorly understood. 7 Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.6 Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.5 Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.4 Maternal-Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.3 Core Center of Excellence in Nano Imaging, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA.2 The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.1 Department of Surgery, The Vision Center, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA. ![]()
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