July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Towards macular regeneration: generation, MACS purification and transplantation of cone photoreceptors derived from mouse induced pluripotent stem cells (mIPSC).
Author Affiliations & Notes
  • Hany Abdelgawad
    Department Of Ophthalmology, Harvard Medical School , Boston, Massachusetts, United States
    Department Of Ophthalmology, Fayoum Univeristy, Fayoum, Egypt
  • Jeayoung Park
    Department Of Ophthalmology, Harvard Medical School , Boston, Massachusetts, United States
    Yale School of Medicine, New Haven, Connecticut, United States
  • Koyar Afrasyab
    Department Of Ophthalmology, Harvard Medical School , Boston, Massachusetts, United States
    Linköping University, Linköping, Sweden
  • Petr Y Baranov
    Department Of Ophthalmology, Harvard Medical School , Boston, Massachusetts, United States
  • Michael J. Young
    Department Of Ophthalmology, Harvard Medical School , Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Hany Abdelgawad, None; Jeayoung Park, None; Koyar Afrasyab, None; Petr Baranov, None; Michael Young, None
  • Footnotes
    Support  Bertarelli Foundation 4100050
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 556. doi:
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      Hany Abdelgawad, Jeayoung Park, Koyar Afrasyab, Petr Y Baranov, Michael J. Young; Towards macular regeneration: generation, MACS purification and transplantation of cone photoreceptors derived from mouse induced pluripotent stem cells (mIPSC).. Invest. Ophthalmol. Vis. Sci. 2018;59(9):556.

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

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Abstract

Purpose : Cone photoreceptors are responsible for color vision and high visual acuity. Cone degenerative disorders such as cone dystrophy are characterized by irreversible loss of these cells. Stem cell replacement therapy is a possible therapeutic strategy for these diseases. Two major obstacles to development of this strategy are the lack of cone surface markers that can be reliably used to enrich the cone population and the low percentage of stem-cell derived cones in the retinal organoids.

Methods : To increase the generated s-opsin cones from CRX-GFP mIPSC, we modified Sasai's protocol of optic cup generation by inhibiting the NOTCH pathway from D12-14 and removing retinoic acid from the medium.
To check the specificity of peanut agglutinin (PNA) Magnetic-Activated Cell Sorting (MACS) system, adult WT mice and rats' retinas were dissociated in single cells, stained with PNA-biotin and sorted using anti-biotin microbeads. Then we applied the same method to retinal organoids at D16 of differentiation. The unsorted, PNA+ and PNA- fractions from the WT retinas and the organoids were analyzed and characterized by flow cytometry, immunohistochemistry and RT-qPCR. The PNA+ fraction from the organoids was subretinally transplanted into the adult WT mice retinas. Two weeks after injection, mice were sacrified and the injected eyes were cryosectioned and double stained for GFP and cone arrestin.

Results : Flow cytometry analysis of retinal organoids at D16 indicated that s-opsin cones represented (56% ± 5%, N = 5, n = 48) of total organoid cells. PNA-MACS method yeiled significant high purity (89% ± 4%, N = 4) and viability (98% ± 0.5%, N = 3).
RT-qPCR analysis showed significant expression of cone-specific genes in PNA+ cells when compared to PNA- or unsorted cells.
The transplanted PNA+ cells from the D16 retinal organoids integrated into the outer nuclear layer of the adult WT mouse retina after 2 weeks of injection and express different mature cones markers such as cone arrestin without evidence of tumor formation.

Conclusions : Cone photoreceptors can be generated in high percentage from the retinal organoids and can be enriched via PNA-MACS method.These promising results make the transplantation of a tissue-engineered macula a pursuable goal and provide a new method to isolate cones for further studies

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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