June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Developing novel differentiation methods for the generation of 3D retinal organoids from cynomolgus macaque iPSCs
Author Affiliations & Notes
  • Madeleine Carter
    Biosciences Institute, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom
  • Valeria Chichagova
    Newcells Biotech, United Kingdom
    Biosciences Institute, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom
  • Birthe Dorgau
    Biosciences Institute, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom
  • Majlinda Lako
    Biosciences Institute, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom
  • Footnotes
    Commercial Relationships   Madeleine Carter None; Valeria Chichagova None; Birthe Dorgau None; Majlinda Lako None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3731 – F0337. doi:
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      Madeleine Carter, Valeria Chichagova, Birthe Dorgau, Majlinda Lako; Developing novel differentiation methods for the generation of 3D retinal organoids from cynomolgus macaque iPSCs. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3731 – F0337.

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

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Abstract

Purpose : To develop a reliable in vitro protocol for generating 3D retinal organoids (ROs) from cynomolgus macaque iPSCs using a highly scalable multi-well plate (wp) format. This aims to inform knowledge of mammalian retinal differentiation and reduce the pre-clinical use of animal models.

Methods : Two iPSC lines from cynomolgus macaque (Macaca fascicularis) were exposed to several known and novel human retinal differentiation protocols, using early BMP4 addition, an IGF1 based media, or RPE conditioned media (-CM), in a 96-wp format. The differentiation protocols were applied using a timeline adjusted for the shorter macaque gestation, relative to human.
iPSC-derived ROs were characterised morphologically and molecularly throughout differentiation, for protein and gene expression of known neural-retinal and mature cell markers. Immunohistochemistry was performed using validated antibodies. RT-qPCR was performed on RNA from multiple organoids, using validated primers. Two-way ANOVA analysis of gene expression data was performed on two independent replicates.

Results : By adapting the timeline of human RO differentiation protocols to the shorter gestation of the macaque, macaque iPSC derived ROs were generated and maintained phase-bright retina and mature cell marker expression at day 120. Macaque ROs show the sequential expression of neural-epithelia (RAX, PAX6, SOX2), photoreceptor precursor (CRX), interneuron (AP2α, PRKCα), photoreceptor (ARR3, OPN1SW, OPN1LW/MW, RHO), and Müller glia (RLBP1) markers indicating some retinal development and maturation throughout differentiation. Method efficiency to generate ROs as determined by morphology was ~40% at day 120.
Macaque iPSC lines showed variable responses, with the IGF1-based protocol improving RO structure and photoreceptor gene expression (RCVRN, ARR3, CRX) significantly (p<0.0001) in both cell lines, and additionally significantly increasing Müller glia (RLBP1) and rod (RHO) expression (p<0.0001) in 1 cell line. RPE-CM addition from day 15 significantly increased Müller glia (RLBP1) and cone (ARR3) expression (p<0.0001) at late stages (day 90-120) in both cell lines.

Conclusions : The use of an adapted differentiation timeline and retinal stimulating factors, IGF1 and RPE-CM, resulted in high method efficiencies for differentiation of cynomolgus macaque iPSCs to ROs and confirms the amenability of production in a multi-well format.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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