Abstract
A major goal in the stem cell field is to generate tissues that can be utilized as a universal tool for in vitro models of development and disease, drug development, or as a resource for patients suffering from disease or injury. Great efforts are being made to differentiate human pluripotent stem cells in vitro toward retinal tissue, which is akin to native human retina in its cytoarchitecture and function, yet the numerous existing retinal induction protocols remain variable in their efficiency and do not routinely produce morphologically or functionally mature photoreceptors. Herein, we determine the impact that the method of embryoid body (EB) formation and maintenance as well as cell line background has on retinal organoid differentiation from human embryonic stem cells and human induced pluripotent stem cells. Our data indicate that cell line‐specific differences dominate the variables that underline the differentiation efficiency in the early stages of differentiation. In contrast, the EB generation method and maintenance conditions determine the later differentiation and maturation of retinal organoids. Of the latter, the mechanical method of EB generation under static conditions, accompanied by media supplementation with Y27632 for the first 48 hours of differentiation, results in the most consistent formation of laminated retinal neuroepithelium containing mature and electrophysiologically responsive photoreceptors. Collectively, our data provide substantive evidence for stage‐specific differences in the ability to give rise to laminated retinae, which is determined by cell line‐specific differences in the early stages of differentiation and EB generation/organoid maintenance methods at later stages.
Highlights
An armory of protocols exist which facilitate the generation of retinal tissue from human pluripotent stem cells
We determine the impact that the method of embryoid body (EB) formation and maintenance as well as cell line background has on retinal organoid differentiation from human embryonic stem cells and human induced pluripotent stem cells
EBs cultured in the absence of Y27632 appeared homogenous and phase-bright during the first 48 hours, whereas ROCK inhibition (ROCKi) resulted in early organization of EBs into neuroepithelial-like formations
Summary
An armory of protocols exist which facilitate the generation of retinal tissue from human pluripotent stem cells. The production of photoreceptors from human stem cells was originally reported under two-dimensional (2D) culture conditions which gave rise to photoreceptor precursor cells and some photoreceptors expressing mature photoreceptor markers [1,2,3,4,5] This was achieved in three-dimensional (3D) culture, which gave rise to developing photoreceptors within optic vesicle-like structures that consisted of multiple retinal progeny [6] within distinct retinal laminae [7,8,9]. This advance enabled the production of retinal tissue, which more closely mimicked the natural order of retinogenesis and microarchitecture of the native retina than. There is a clear need for a greater understanding of the events controlling retinal organoid formation in order to improve upon and standardize protocols for the routine orchestration of useful retinal tissue across multiple cell lines
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