July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Generation of rod- vs. cone-dominant patient-derived 3D retinal grafts for the treatment of retinal degenerative blindness
Author Affiliations & Notes
  • Laura R. Bohrer
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Jessica A. Cooke
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Arwin Shrestha
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
    Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, United States
  • Erin R. Burnight
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Kristin R. Anfinson
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Malia M. Collins
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Robert F Mullins
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Edwin M Stone
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Kristan S. Worthington
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
    Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, United States
  • Luke A. Wiley
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Budd Tucker
    Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Laura Bohrer, None; Jessica Cooke, None; Arwin Shrestha, None; Erin Burnight, None; Kristin Anfinson, None; Malia Collins, None; Robert Mullins, None; Edwin Stone, None; Kristan Worthington, None; Luke Wiley, None; Budd Tucker, None
  • Footnotes
    Support  R01EY024588; R01EY026008, R01EY026087
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2871. doi:
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      Laura R. Bohrer, Jessica A. Cooke, Arwin Shrestha, Erin R. Burnight, Kristin R. Anfinson, Malia M. Collins, Robert F Mullins, Edwin M Stone, Kristan S. Worthington, Luke A. Wiley, Budd Tucker; Generation of rod- vs. cone-dominant patient-derived 3D retinal grafts for the treatment of retinal degenerative blindness. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2871.

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

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Abstract

Purpose : Induced pluripotent stem cell (iPSC)-derived photoreceptor cells hold great promise for restoring vision to patients with retinal degenerative blindness. Unfortunately, traditional cell isolation and subretinal transplantation approaches result in poor donor cell survival. More sophisticated tissue engineering-based strategies, aimed at reducing post-transplant cell death by providing cells as a contiguous graft, are more promising. The purpose of this study was to combine CRISPR-based genome editing and iPSCs generated from patients with enhanced S-cone syndrome to develop rod- vs. cone-dominant photoreceptor cell grafts for the purpose of evaluating the effects of developmental age on photoreceptor cell axon extension and synapse formation.

Methods : IPSCs were generated using dermal fibroblasts isolated from 2 patients with molecularly confirmed NR2E3-associated enhanced S-cone syndrome. CRISPR-Cas9-based homology-dependent repair strategies were designed to correct NR2E3. Retinal organoids were derived using a stepwise 3D differentiation protocol. Organoids were dissociated and seeded in polycaprolactone (PCL) scaffolds generated using two-photon lithography. Retinal cell grafts were analyzed via immunocytochemistry.

Results : To generate cone-dominant grafts, iPSCs from 2 patients (P1 – IVS1/IVS1, P2 – R73/R311) with NR2E3-associated enhanced S-cone syndrome were used. Throughout early stages of development organoids generated from these patients formed polarized neural epithelium expressing PAX6 followed by OTX2 and RORb. In comparison to normal controls, which at 95-120 days post-differentiation contained <1% S-opsin positive cone photoreceptor precursor cells, 10-15% of the cells contained within NR2E3 patient-derived organoids were S-opsin positive (P<0.01). To restore rod generation potential, and allow us to generate rod- vs. cone-dominant grafts from the same individuals, NR2E3 mutations were corrected via CRISPR-Cas9-based homology-dependent repair. 3D PCL cell delivery scaffolds were seeded with retinal progenitor cells isolated from iPSC-derived retinal organoids. At 1-week post-seeding, grafts were densely populated with OTX2-positive photoreceptor precursor cells.

Conclusions : By combining CRISPR-based genome editing and patient-derived iPSCs, we can generate rod- vs. cone-dominant grafts suitable for analysis of cell type specific regenerative capacity.

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

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