June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Development of a novel iPSC-derived 3D-retinal organoid model system from cone-dominant Tree Shrews
Author Affiliations & Notes
  • Sangeetha Kandoi
    Ophthalmology, University of California San Francisco, San Francisco, California, United States
  • Monichan Hayes Phay
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, US, academic/medres, Boston, Massachusetts, United States
    Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Cassandra Martinez
    Ophthalmology, University of California San Francisco, San Francisco, California, United States
  • Petr Y Baranov
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, US, academic/medres, Boston, Massachusetts, United States
    Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Deepak A Lamba
    Ophthalmology, University of California San Francisco, San Francisco, California, United States
  • Footnotes
    Commercial Relationships   Sangeetha Kandoi, None; Monichan Phay, None; Cassandra Martinez, None; Petr Baranov, None; Deepak Lamba, None
  • Footnotes
    Support  U24EY029891, P30 Vision Core Grant, RPB unrestricted funds
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 3152. doi:
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      Sangeetha Kandoi, Monichan Hayes Phay, Cassandra Martinez, Petr Y Baranov, Deepak A Lamba; Development of a novel iPSC-derived 3D-retinal organoid model system from cone-dominant Tree Shrews. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3152.

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

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Abstract

Purpose : Visual dysfunctions affecting the central retina including highly prevalent age-related macular degeneration occurs due to degeneration of the cone-dominant central macula region leading to high-acuity vision loss. Unfortunately, there is a lack of an easily accessible animal model that resembles the human retinal macula. Tupaia belangeri (Tree Shrews, Ts) exhibit an extremely high cone: rod ratio (~95%), have large eyes with a more human-like lens: globe ratio, and exhibit highly visual behaviors. Hence the goal of this study was to develop a novel stem cell-based 3D retina from the diurnal, cone-dominant, non-rodent primate-like, Ts.

Methods : Neural progenitor cells (NPC) obtained from a neonatal male, Ts (Gift by Brian Samuels, UAB) was used to generate a stable induced pluripotent stem cell (iPSC) line using CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit. Individual iPSC clones were expanded and assessed for pluripotency markers via immunofluorescence (IF). 3D-retinal differentiation was carried out using a three-step transitioning protocol (3D-2D-3D) with neural induction media. Retinal organoids (RO) collected at various time-points of differentiation (Day 25, 40, 60) were evaluated for retinal cell types by IF and qRT-PCR, and phenotypically compared with neonatal Ts eyes.

Results : iPSC lines (n=3 clones) generated from Ts-NPCs exhibited typical morphological characteristics: compact colonies with clear borders and high nucleus: cytoplasm ratio by bright field microscopy. IF confirmed the expression of pluripotent markers - Oct3/4, Sox2, and Nanog. Undifferentiated Ts-iPSCs directed towards retinal fate generated self-assembled laminated 3D-RO. Protein and gene expression analysis confirmed markers of multipotent retinal progenitor cells (Lhx2, Pax6, Sox2), ganglion/amacrine cells (Brn3a, Islet, HuC/D), and photoreceptors (Otx2, Blimp1, Crx, Rcvrn).

Conclusions : This is the first study to demonstrate the establishment of iPSCs from a novel primate-like tree shrew with cone-dependent visual ecology. Additionally, we have successfully developed a method to generate photoreceptors containing 3D-RO. This approach has a great potential to both understand the feasibility of cone photoreceptor replacement and facilitate the development of novel cone-degeneration models in vitro for understanding and ameliorating catastrophic vision loss using this clinically relevant primate-like animal model.

This is a 2021 ARVO Annual Meeting abstract.

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