July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Variation in retinal differentiation capacity between patient derived hiPSC lines.Variation in retinal differentiation capacity between patient derived hiPSC lines.
Author Affiliations & Notes
  • Jessica A Cooke
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Cathryn M Cranston
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Elizabeth L Kennedy
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Adam Deluca
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • S. Scott whitmore
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Todd E Scheetz
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Robert F Mullins
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Edwin M Stone
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Budd A Tucker
    Institute for Vision Research, Ophthalmology and Visual Science, The University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Jessica Cooke, None; Cathryn Cranston, None; Elizabeth Kennedy, None; Adam Deluca, None; S. whitmore, None; Todd Scheetz, None; Robert Mullins, None; Edwin Stone, None; Budd Tucker, None
  • Footnotes
    Support  NIH Grants R01EY024588; R01EY026008, R01EY026087
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2874. doi:
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      Jessica A Cooke, Cathryn M Cranston, Elizabeth L Kennedy, Adam Deluca, S. Scott whitmore, Todd E Scheetz, Robert F Mullins, Edwin M Stone, Budd A Tucker; Variation in retinal differentiation capacity between patient derived hiPSC lines.Variation in retinal differentiation capacity between patient derived hiPSC lines.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2874.

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

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Abstract

Purpose : Human induced pluripotent stem cells (hiPSCs) can be differentiated into a variety of different cell types. Protocols focused on 3D neural retinal differentiation generally give rise to two distinct populations, retina and forebrain. That said, there is considerable variability between patient derived iPSC lines in the frequency of retinal organoid production. The purpose of this study was to identify distinct gene expression profiles between hiPSC lines associated with low vs high percentage retinal organoid production.

Methods : HiPSCs were generated using dermal fibroblasts via Sendai virus driven expression of OCT4, SOX2, KLF4 and C-MYC. hiPSC lines were validated via score card and karyotype analysis and differentiated toward a retinal cell lineage using a stepwise 3D differentiation protocol. Samples were taken at four time points throughout the first twenty days of differentiation. Retinal differentiation capacity was determined by qRT-PCR and ICC analysis. RNA was isolated and RNASeq analysis was performed. Differential gene expression profiles were compared between lines with low vs high retinal differentiation capacity.

Results : Biopsies were obtained and dermal fibroblasts were isolated from 15 individuals, including normal controls and those with a variety of ophthalmic diseases. HiPSCs were generated and validated cell lines were differentiated into 3D retinal organoids via stepwise differentiation. At 20 days post-differentiation, cultures were microscopically evaluated. HiPSCs from 9 individuals were determined to be good retinal producers (i.e., 40-80% of the total 3D organoids being of retinal lineage) while lines from the remaining 7 individuals were determined to have a low retinal yield (i.e., <30% of organoids being of retinal lineage). No correlation between disease causing genotype and retinal production yield was identified. RNAseq analysis focused on identifying genes differentially expressed between these two populations pre-differentiation is currently underway.

Conclusions : In conclusion approximately 55% of all hiPSC lines produced, regardless of genetic background, have the potential to differentiate into retinal organoids with high yield. Identification of a genetic signature specifically associated with ability to adopt a retinal cell fate may be useful for pre-differentiation hiPSC screening.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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