Systematic Comparison of Retinal Organoid Differentiation from Human Pluripotent Stem Cells Reveals Stage Specific, Cell Line, and Methodological Differences

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.

Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective

“…Many teams are focusing on new methodologies to improve the quality of retinal organoids by tweaking protocols, using bioreactors, or microfluidic systems, [163,167,169,170]. Recently, Mellough et al (also with slight modification performed by Cowan et al) showed that mechanical dissociation at the embryoid body formation stage lead to improved formation of human retinal organoids [208,209]. The use of bioreactor setups, instead of static culture setups, may help solve a number of these problems as they allow for improved aeration and distribution of nutrients as well as allow for scaling up of organoid production.…”

Limitations and Promise of Retinal Tissue From Human Pluripotent Stem Cells for Developing Therapies of Blindness

“…Variability in size, shape, photoreceptor density, lamination determined by cell line-specific and protocol-specific differences (both derivation and maintenance) were described and documented ( Capowski et al, 2019 ; Cowan et al, 2019 ; Mellough et al, 2019 ). However, it is feasible even with current technologies to culture and maintain organoids for longer than one year, as a proof-of-principle ( Capowski et al, 2019 ).…”

Retinal and Brain Organoids: Bridging the Gap Between in vivo Physiology and in vitro Micro-Physiology for the Study of Alzheimer’s Diseases

“…Similarly to wholebrain organoids, culturing iPSC-derived EBs in predetermined conditions generates 3D optic cup-like structures together with stratified neural retinal cell subtypes and progenitors. After this seminal work, several protocols have been proposed to drive the differentiation of human iPSC toward retinal organoids (Mellough et al, 2019). Current retinal differentiation protocols can produce most relevant retinal cell types in laminated fashion with a variable efficiency in generating functional photoreceptors (Zhong et al, 2014) to retinal ganglion cells (Ohlemacher et al, 2016), and overall mature retinal tissues.…”