Abstract
Purpose :
Stem cell-derived retinal organoids (ROs) facilitate investigations into retinal development, transplantation therapies, and disease modeling. While RO transplantation may hold the potential to restore vision in patients with retinal disease, its translational use is currently impeded due to heterogeneity among ROs presumably resulting from the differences in culturing protocols. To develop safe and effective clinical treatment involving the augmentation of ROs to restore visual function, it is crucial to address the variations in cellular and molecular composition of ROs cultured using different protocols. In this study, we investigate the influence of different culturing protocols on the composition of retinal organoids.
Methods :
We used Single-cell and single nuclei RNA sequencing (scRNA-seq and snRNA-seq) published datasets of ROs (GSE142526, GSE201356, and GSE183684) each generated using its unique culturing protocol. In addition, we also generated an in-house dataset of cultured retinal organoids. The organoids were dissociated into a single-cell suspension and run on the 10X Genomics Chromium single-cell platform. Raw data was processed using the 10X Genomics cellranger count and analyzed using the Seurat 5.0 R package. The combined data set underwent normalization, variable feature selection, and scaling using the default parameters in Seurat. Batch removal and dataset integration were performed using Harmony. Uniform Manifold Approximation and Projection (UMAP) dimension reduction was performed on the top 20 corrected principal components, and clusters were computed. Cell types were then identified using a list of known marker genes.
Results :
Divergent culturing protocols for retinal organoids yield unique and heterogeneous cell type populations, with specific protocols favoring the prevalence of distinct cell types. The presence of astrocytes and Brain and spinal cord-like cells (BSLCs) in cultured retinal organoids contrasts with their absence in established retinal development datasets.
Conclusions :
Further inquiries are imperative to refine the precision of retinal organoid specification and cultivation, aiming to alleviate the emergence of these undesirable cell types. The development of organoids that closely mimic the developing human retina could help advance therapeutic development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.