June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Enhanced reproducibility of retinal organoids enables the analysis of early human retinal fate specification
Author Affiliations & Notes
  • Jade Harkin
    Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States
    Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Kiersten Pena
    Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana, United States
  • Sailee Sham Lavekar
    Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana, United States
    Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Elyse Feder
    Medicine, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Kelly Lentsch
    Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana, United States
  • Jason S Meyer
    Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, United States
    Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Footnotes
    Commercial Relationships   Jade Harkin None; Kiersten Pena None; Sailee Lavekar None; Elyse Feder None; Kelly Lentsch None; Jason Meyer Wisconsin Alumni Research Foundation, Code P (Patent)
  • Footnotes
    Support  NIH R01EY024984, NIH R01EY033022, NIH U24EY033269, BrightFocus Foundation G2020369, Glaucoma Research Foundation
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3728 – F0334. doi:
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    • Get Citation

      Jade Harkin, Kiersten Pena, Sailee Sham Lavekar, Elyse Feder, Kelly Lentsch, Jason S Meyer; Enhanced reproducibility of retinal organoids enables the analysis of early human retinal fate specification. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3728 – F0334.

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

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Abstract

Purpose :
Retinal organoids can be differentiated from human pluripotent stem cells (hPSCs) that effectively recapitulate the major stages of human retinogenesis. These organoids are becoming valuable tools for studying human retinogenesis and retinal diseases, yet shortcomings in the efficiency and reproducibility of current retinal organoid differentiation protocols have hindered their abilty to serve as effective models for the earliest stages of human retinal lineage specification.

Methods : In the current study, we refined an existing retinal organoid differentiation protocol using more standardized, quick reaggregation methods to generate highly reproducible 3D retinal organoids from human pluripotent stem cells (hPSCs). BMP signaling contributing to retinal specification was analyzed by treatment with either BMP4 or the BMP inhibitor LDN-193189, and differentiation efficiency was assessed at various time points based on morphological analyses and the expression of retinal markers. Additionally, to identify transcriptional changes that underly retinal fate dermination events, mRNA-seq analyses were conducted at the earliest stages of retinal specification.

Results :
Retinal organoids generated using quick reaggregation methods were highly reproducible in both their size and shape compared to more traditional methods. Following treatment of early aggregates with either BMP4 or LDN-193189, pure populations of either retinal or forebrain organoids were derived, respectively. Subsequently, RNA-seq methods analyzed the transcriptional profile of the earlies stages of retinal vs forebrain specification, long before these lineages have been reliably identified previously. These refined methods also yielded retinal organoids with greatly expedited differentiation timelines, with differentiated retinal neurons arising at earlier stages than traditional differentiation methods, also exhibiting higher levels of self-organization.

Conclusions : Taken together, this study provides a novel and highly reproducible method for generating retinal organoids suitable for analyzing the earliest stages of human retinal fate specification in an organoid model. These results elucidate some of the earliest transcriptional changes occurring at the most immediate stages of human retinal development, and provide a more optimized and rapid method for generating retinal organoids for translational applications.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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