Investigative Ophthalmology & Visual Science Cover Image for Volume 61, Issue 7
June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Characterization and staging of outer plexiform layer morphogenesis and synaptogenesis in human retinal organoids
Sumitha Prameela Bharathan; Angela Ferrario; Kayla Stepanian; Rosanna Calderon; G. Esteban Fernandez; mark reid; Carolyn Marks; Matthew Thornton; Brendan Grubbs; Thomas Lee; David Cobrinik; Jennifer Aparicio; Aaron Nagiel
Author Affiliations & Notes
  • Sumitha Prameela Bharathan
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Angela Ferrario
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Kayla Stepanian
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Rosanna Calderon
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    Development, Stem Cell and Regenerative Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • G. Esteban Fernandez
    The Cellular Imaging Core, The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, United States
  • mark reid
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Carolyn Marks
    The Core Centre for Excellence in Nano Imaging, USC Viterbi School of Engineering of the University of Southern California, Los Angeles, California, United States
  • Matthew Thornton
    Maternal-Fetal Medicine Division of the Department of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Brendan Grubbs
    Maternal-Fetal Medicine Division of the Department of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Thomas Lee
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • David Cobrinik
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Jennifer Aparicio
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Aaron Nagiel
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Sumitha Prameela Bharathan, None; Angela Ferrario, None; Kayla Stepanian, None; Rosanna Calderon, None; G. Esteban Fernandez, None; mark reid, None; Carolyn Marks, None; Matthew Thornton, None; Brendan Grubbs, None; Thomas Lee, None; David Cobrinik, None; Jennifer Aparicio, None; Aaron Nagiel, None
  • Footnotes
    Support  Research to Prevent Blindness, New York; The Las Madrinas Endowment in Experimental Therapeutics for Ophthalmology; The Knights Templar Eye Foundation; The Baxter Foundation
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4015. doi:
  • Views
  • Share
  • Tools
    • Alerts
      User Alerts
      You are adding an alert for:
      Characterization and staging of outer plexiform layer morphogenesis and synaptogenesis in human retinal organoids
      You will receive an email whenever this article is corrected, updated, or cited in the literature. You can manage this and all other alerts in My Account
      The alert will be sent to:
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation
      Citation

      Sumitha Prameela Bharathan, Angela Ferrario, Kayla Stepanian, Rosanna Calderon, G. Esteban Fernandez, mark reid, Carolyn Marks, Matthew Thornton, Brendan Grubbs, Thomas Lee, David Cobrinik, Jennifer Aparicio, Aaron Nagiel; Characterization and staging of outer plexiform layer morphogenesis and synaptogenesis in human retinal organoids. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4015.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

      ×
    • Get Permissions
  • Supplements
Abstract

Purpose : The first synapse of the visual system between photoreceptors and bipolar cells is critical for visual processing, and its formation at the outer plexiform layer (OPL) requires a complex orchestration of cellular and molecular events. This study seeks to establish the human retinal organoid (HRO) system to elucidate human photoreceptor-bipolar cell synapse formation, specificity, and maintenance during retinal development and disease.

Methods : HROs were generated from human induced pluripotent stem cells using published protocols and sampled from day 100 in culture (D100) to D280. Confocal immunofluorescence (IF) analysis of fixed sections was performed to identify the spatiotemporal organization of retinal cell types and the subcellular localization of ribbon synapse proteins.

Results : IF analysis of HRO sections with cell type-specific markers for cones, rods, bipolar cells and horizontal cells revealed a window from D100 to D160 during which retinal cell types organized and matured to form an OPL. However, extensive inter- and intra-organoid variability in HRO maturation necessitated a staging system for OPL development. Human fetal retina sections were thus studied to define a reliable staging system based on cellular architecture. Five distinct morphogenetic stages (stage 0 to 4) were identified in the retina at fetal week 16.6. We found that stages 0 to 3, but not stage 4 were reflected in our HROs. To confirm the functional relevance of this staging system, we demonstrate increasingly precise subcellular localization of ribbon synapse proteins such as Bassoon (p<0.0001) from stage 0 to 3 in the HROs. Regions of stage 3 OPL in HROs contain the expected juxtaposition of ribbon synapse proteins and the postsynaptic ON-bipolar receptor mGluR6.

Conclusions : Through correlative analysis of human fetal retina, we define key stages of OPL development and set the stage to use HROs for study of human photoreceptor-bipolar cell synaptogenesis during development, in the disease state, and following treatment with gene or cellular therapies.

This is a 2020 ARVO Annual Meeting abstract.

 

Recapitulation of outer retinal development by HRO with laminar organization of cones (ARR3), rods (RHO), rod bipolar cells (PKCα), ON-bipolar cells (PCP2) and horizontal cells (CALB).
View OriginalDownload Slide

Recapitulation of outer retinal development by HRO with laminar organization of cones (ARR3), rods (RHO), rod bipolar cells (PKCα), ON-bipolar cells (PCP2) and horizontal cells (CALB).

Recapitulation of outer retinal development by HRO with laminar organization of cones (ARR3), rods (RHO), rod bipolar cells (PKCα), ON-bipolar cells (PCP2) and horizontal cells (CALB).

Recapitulation of outer retinal development by HRO with laminar organization of cones (ARR3), rods (RHO), rod bipolar cells (PKCα), ON-bipolar cells (PCP2) and horizontal cells (CALB).
View OriginalDownload Slide
 
Colocalization of ribbon synapse proteins Bassoon and Ribeye at the OPL in a stage 3 HRO.
View OriginalDownload Slide

Colocalization of ribbon synapse proteins Bassoon and Ribeye at the OPL in a stage 3 HRO.

Colocalization of ribbon synapse proteins Bassoon and Ribeye at the OPL in a stage 3 HRO.

Colocalization of ribbon synapse proteins Bassoon and Ribeye at the OPL in a stage 3 HRO.
View OriginalDownload Slide

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Attribution-NonCommercial-NoDerivatives
Copyright © 2025 Association for Research in Vision and Ophthalmology.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×
This site uses cookies. By continuing to use our website, you are agreeing to our privacy policy.Accept