Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines

Abstract

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent

“…Despite the ability of these protocols to give rise to laminated retinal organoids, variability in the propensity of iPSCs to give rise to various retinal cell types is often reported. Several groups including ours have reported variable laminar organization between samples differentiated from the same iPSC lines and the presence of non‐neural cell types alongside the retinal structures …”

“…Several groups including ours have reported variable laminar organization between samples differentiated from the same iPSC lines and the presence of non-neural cell types alongside the retinal structures. 7,11,12 Retinal development in vivo is controlled by a diverse set of signaling pathways and complex interactions between embryological tissues which affect the identity of the resultant cell population.…”

Deciphering retinal diseases through the generation of three dimensional stem cell-derived organoids: Concise Review

“…Zhong et al , Parfitt et al , Wahlin et al , and others recently described the examples of organoid photoreceptor outer segments. In their studies, the electron microscopy imaging revealed other ultrastructural features of mature photoreceptors, including an outer limiting membrane, inner segments rich in mitochondria, and basal bodies with connecting cilia displaying a photoreceptor‐specific microtubule arrangement.…”

“…As previously mentioned, a number of described protocols have permitted the generation of retinal tissue from hPSCs with varying efficiencies allowing us to mimic the early stages of human eye development and to model human retinal disease pathogenesis and repair [15,17,19,[21][22][23][24][25][26][27] (Tables 1 and 2). These stratified structures display some human-specific features: they are larger in size compared with mouse counterparts and contain different retinal structures, including an outer nuclear layer with photoreceptors (rhodopsin+, recoverin+), an inner nuclear layer with horizontal (calbindin+, calretinin−), bipolar (Chx10+, Pax6−), amacrine (Pax6+, calretinin+), Müller glia cells (CRALBP+), and a RGC layer with RGCs (Brn3+, Pax6+, calretinin+) ( Fig.…”

The Emergence of Stem Cell‐Based Brain Organoids: Trends and Challenges

“…The pioneering work of the Sasai laboratory has uncovered these astonishing properties of pluripotent cells . Although culturing retinal organoids consumes substantial amount of time up to 150 days, several laboratories have successfully generated optic cups or retinal organoids harboring retinal pigment epithelia and functional photoreceptors …”