Retinal organoids generated from rhesus macaque (Macaca Mulatta) induced pluripotent stem cells demonstrate mature photoreceptors and rods

Abstract

Purpose : To characterize the development of non-human primate (NHP) derived retinal organoids in comparison to those derived from human embryonic stem cells (hESCs).

Methods : Human embryonic stem cells (hESCs) and induced-pluripotent stem cells (rhiPSCs) derived from non-human primates (Macaca mulatta) were differentiated into retinal organoids by using a well-established differentiation protocol. Briefly, embryoid bodies were formed from pluripotent stem cells and induced into neural lineage with neural induction media with the addition of BMP4. Thereafter, self-formation of optic vesicles was allowed to form in a 2D culture in retinal differentiation media (RDM). Optic vesicles were then picked off and suspended in 3D-RDM until analysis. Differences in the timing of differentiation and efficiency of retinal organoid development were assessed by light microscopy, electron microscopy, immunocytochemistry, and next-generation sequencing.

Results : Generation of retinal organoids was achieved from both human and several NHP pluripotent stem cells lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had defined structure and were composed of mature retinal cells types including cone and rod photoreceptors. Characterization of cell populations in NHP retinal organoids were assessed by single cell RNA sequencing which allowed comparison of expression profiles of retinal neurons to hESC derived retinal organoids and to samples of retinal tissue. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While culture of NHP-derived iPSCs is relatively difficult compared to human stem cells, generation of retinal organoids is feasible and may be more time effective due to an intrinsically faster timing of retinal differentiation.

Conclusions : Retinal organoids can be produced from iPSCs derived from rhesus macaque using established protocols. Important species-specific differences exist that warrant further investigation. The production of NHP retinal organoids may be advantageous to reduce experimental time and cost for basic biology in retinogenesis as well as for preclinical trials in NHPs.

This is a 2021 ARVO Annual Meeting abstract.