June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Propensity of patient-derived hiPSCs for retinal differentiation
Author Affiliations & Notes
  • Jessica A Cooke
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Andrew P Voigt
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Michael A Collingwood
    Enzyme Evolution, Integrated DNA Technologies Inc, Coralville, Iowa, United States
  • Nicholas Edwin Stone
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • S Scott Whitmore
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Adam P DeLuca
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Kristin R. Anfinson
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Christopher A Vakulskas
    Enzyme Evolution, Integrated DNA Technologies Inc, Coralville, Iowa, United States
  • Austin J Reutzel
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Heather T Daggett
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Jeanean L Andorf
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Edwin M Stone
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Robert F Mullins
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Budd A. Tucker
    Institute for Vision Research, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Jessica Cooke None; Andrew Voigt None; Michael Collingwood Integrated DNA Technologies, Code E (Employment); Nicholas Stone None; S Scott Whitmore None; Adam DeLuca None; Kristin Anfinson None; Christopher Vakulskas Integrated DNA Technologies, Code E (Employment); Austin Reutzel None; Heather Daggett None; Jeanean Andorf None; Edwin Stone None; Robert Mullins None; Budd Tucker None
  • Footnotes
    Support  Institute for Vision Research, Ruby Endowed Chair of Regenerative Ophthalmology
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3730 – F0336. doi:
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    • Get Citation

      Jessica A Cooke, Andrew P Voigt, Michael A Collingwood, Nicholas Edwin Stone, S Scott Whitmore, Adam P DeLuca, Kristin R. Anfinson, Christopher A Vakulskas, Austin J Reutzel, Heather T Daggett, Jeanean L Andorf, Edwin M Stone, Robert F Mullins, Budd A. Tucker; Propensity of patient-derived hiPSCs for retinal differentiation. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3730 – F0336.

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

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Abstract

Purpose : To identify a transcriptomic profile in undifferentiated and early differentiating hiPSCs that would predict a given cell line’s propensity to generate retinal cells.

Methods : Human iPSCs generated via Sendai virus were validated by scorecard and karyotype analysis prior to differentiation. hiPSC lines were differentiated toward a retinal cell lineage using a stepwise 3D differentiation protocol. RNA was isolated from days 0 and 7 of differentiation and subjected to RNA-sequencing. Transcriptome profiles were determined and validated from a total of 15 independent donors with low vs. high retinal differentiation capacity. Findings were validated in a masked fashion using an additional 16 cell lines.

Results : At 0 and 7 days post-differentiation, cultures were evaluated using RNAseq. Retinal structures were quantified for retinal phenotype microscopically at 16 and 20 days of differentiation. Analysis was focused on identifying genes differentially expressed between good retinal producers (40-80% of organoids showing a retinal phenotype, n=8) and poor retinal producers (<30% of organoids showing retinal phenotype, n =7). Good and poor retinal producer populations demonstrated remarkable gene expression similarity across all lines prior to differentiation (day 0), which was validated via qPCR-based expression analysis. By just 7 days of differentiation, when embryoid bodies all looked the same, significant differences in gene expression could be detected between good vs poor retinal producers. Ingenuity pathway analysis revealed perturbations in both OCT4 and SOX2 effector gene expression, with expression of many of the factors being maintained in poor producers indicating delayed suppression of pluripotency. This pattern of altered genes expression was validated in an additional 16 cell lines in a masked fashion.

Conclusions : There were no discernable differences in pluripotency or other gene expression in undifferentiated hiPSCs. By day 7 of differentiation, a signature gene expression profile, which predicts a given hiPSC line’s propensity to generate retinal cells upon further differentiation, could be detected. Specifically, 25+ genes that are differentially expressed between days 0 and day 7 of differentiation were identified. Modulation of these key factors may assist in enhancement of retinal differentiation.

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

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