Differentiation methods impact the survival and lamination of human pluripotent stem cell-derived retinal organoids

Abstract

Purpose : The ability to generate three-dimensional (3D) retinal organoids from human pluripotent stem cells (hPSCs) has facilitated the study of developing human retina in vitro, yet the protocols which exist remain variable in their efficiency and are not able to produce functionally mature retinal tissue. We investigated the impact of the method of embryoid body (EB) formation on the retinal differentiation potential of hPSCs. This work is aimed at establishing robust methods to yield functional retinal organoids which mimic the structure and physiology of human retina and thus have broader potential for ophthalmic research.

Methods : Three hPSC lines were differentiated by initiating EB formation followed by long term (150 days) retinal differentiation culture. EBs were generated mechanically, enzymatically or by dissociation-reaggregation in the presence or absence of ROCK inhibitor, then differentiated in parallel under static or shaking conditions. The resulting effect on retinal neuroepithelial formation was determined by in vitro quantification, immunohistochemistry, qRT-PCR, RNA-seq, TEM and electrophysiology.

Results : Utilising a mechanical method of EB formation with ROCK inhibition was consistently the most efficient method of retinal differentiation, generating cultures which gave rise to the greatest quantity of 3D retinal neuroepithelium and optic cups with retinal pigmented epithelium. Mechanically-generated organoids remained viable over long-term culture, developed retinal laminae and gave rise to photoreceptors which developed rudimentary outer segments and retinal ganglion cells which were electrophysiologically active. Differential expression analysis of high-throughput qRT-PCR array data clustered this group of organoids more closely with native human retinal tissue than retinal neuroepithelium generated by other methods. GO ontology analysis of RNA-seq data on day 45 vs 90 highlighted phototransduction as the key enriched signalling pathway.

Conclusions : Our results show that the method of EB formation employed at the onset of differentiation and culture under static vs shaking conditions greatly influence retinal differentiation, organoid viability and the maturity of the retinal progeny. This works towards the development of a functionally improved, more broadly applicable in vitro platform that can be utilised to study eye development and retinal disease.

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