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.