June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Investigating the function of BLIMP1 on bipolar and photoreceptor cell development in human retinal organoids
Author Affiliations & Notes
  • Yen Yu Chang
    Johns Hopkins University, Baltimore, Maryland, United States
  • Casey J. Keuthan
    Johns Hopkins University, Baltimore, Maryland, United States
  • Claire Bell
    Johns Hopkins University, Baltimore, Maryland, United States
  • Xitiz Chamling
    Johns Hopkins University, Baltimore, Maryland, United States
  • Ah Yang
    Johns Hopkins University, Baltimore, Maryland, United States
  • Cynthia A Berlinicke
    Johns Hopkins University, Baltimore, Maryland, United States
  • Donald J Zack
    Johns Hopkins University, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Yen Yu Chang None; Casey Keuthan None; Claire Bell None; Xitiz Chamling None; Ah Yang None; Cynthia Berlinicke None; Donald Zack None
  • Footnotes
    Support  National Eye Institute, Research to Prevent Blindness, Edward N. & Della L. Thome Memorial Foundation, Guerrieri Family Foundation, Foundation Fighting Blindness
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4494. doi:
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      Yen Yu Chang, Casey J. Keuthan, Claire Bell, Xitiz Chamling, Ah Yang, Cynthia A Berlinicke, Donald J Zack; Investigating the function of BLIMP1 on bipolar and photoreceptor cell development in human retinal organoids. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4494.

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

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Abstract

Purpose : In mice, the transcription factor BLIMP1 (Prdm1) has been shown to regulate the cell fate decision that determines whether a progenitor becomes a rod photoreceptor or a bipolar cell. More specifically, BLIMP1 has been reported to increase bipolar cell populations at the expense of photoreceptor populations in mouse models. In order to expand these findings to the human retina and to further explore the molecular basis of BLIMP1’s activity, we generated and are characterizing the effect of BLIMP1 mutations on retinal development in human retinal organoids (hROs).

Methods : CRISPR Cas9 genome editing was used to introduce BLIMP1 mutations into human embryonic stem cell lines (ESCs) carrying CRX/tdTomato and VSX2/EGFP knock-in reporters, and the resulting cells were differentiated into 3D retinal organoids using established protocols. Gene expression analysis was performed by qPCR at different time points throughout differentiation to distinguish the population changes of different retinal cell types. IHC was performed to examine the protein level changes in affected cell types. Single cell transcriptomic analysis is being performed using published Drop-seq protocols.

Results : BLIMP1-/- hROs showed reduced numbers of photoreceptor, both rods and cones, and decreased PR gene expression (e.g., CRX, RCVRN, OPN1SW and RHO), while gene expression of other cell types, including retinal progenitors (e.g., PAX6 and VSX2), RGCs (e.g., BRNB3B and RBPMS) as well as amacrine and horizontal cells (e.g., PROX1 and TFAP2A), was not significantly affected. Although bipolar cell gene expression was significantly altered by BLIMP1 mutation, not all bipolar genes showed increased expression. Only a subset of bipolar cell markers (e.g. VSX1) showed increased expression, while other markers (GRM6 and PRKCA) were decreased.

Conclusions : BLIMP1 mutation leads to changes in the differentiation of photoreceptors and bipolar cells during hRO development, with an overall decrease in photoreceptor populations and an increase of only a subset of bipolar gene markers. Single-cell RNA-seq is underway to identify the subtypes of bipolar cells affected by BLIMP1 mutation. The mechanism of how BLIMP1 contributes to different responses from various subtypes of bipolar cells will be further investigated. This study helps provide a better insight into the development of human bipolar cells.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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