Abstract
Purpose :
Retinal organoids can be differentiated from human pluripotent stem cells (hPSCs) that effectively recapitulate the major stages of human retinogenesis. These organoids are becoming valuable tools for studying human retinogenesis and retinal diseases, yet shortcomings in the efficiency and reproducibility of current retinal organoid differentiation protocols have hindered their abilty to serve as effective models for the earliest stages of human retinal lineage specification.
Methods :
In the current study, we refined an existing retinal organoid differentiation protocol using more standardized, quick reaggregation methods to generate highly reproducible 3D retinal organoids from human pluripotent stem cells (hPSCs). BMP signaling contributing to retinal specification was analyzed by treatment with either BMP4 or the BMP inhibitor LDN-193189, and differentiation efficiency was assessed at various time points based on morphological analyses and the expression of retinal markers. Additionally, to identify transcriptional changes that underly retinal fate dermination events, mRNA-seq analyses were conducted at the earliest stages of retinal specification.
Results :
Retinal organoids generated using quick reaggregation methods were highly reproducible in both their size and shape compared to more traditional methods. Following treatment of early aggregates with either BMP4 or LDN-193189, pure populations of either retinal or forebrain organoids were derived, respectively. Subsequently, RNA-seq methods analyzed the transcriptional profile of the earlies stages of retinal vs forebrain specification, long before these lineages have been reliably identified previously. These refined methods also yielded retinal organoids with greatly expedited differentiation timelines, with differentiated retinal neurons arising at earlier stages than traditional differentiation methods, also exhibiting higher levels of self-organization.
Conclusions :
Taken together, this study provides a novel and highly reproducible method for generating retinal organoids suitable for analyzing the earliest stages of human retinal fate specification in an organoid model. These results elucidate some of the earliest transcriptional changes occurring at the most immediate stages of human retinal development, and provide a more optimized and rapid method for generating retinal organoids for translational applications.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.