High-resolution cell atlas captures human retina in unprecedented details

Use of 4i technology

The human retina organoids that the researchers created from stem cells were the subject of their method. Iterative indirect immunofluorescence imaging, or 4i technology, was at the core of the techniques the researchers employed. Fluorescence microscopy is used in this imaging method to see dozens of proteins in a tiny tissue segment in high resolution. Typically, three proteins in tissue are highlighted with a distinct fluorescent dye using fluorescence microscopy by researchers. 

In contrast, the 3 dyes employed in 4i technology are removed from the tissue sample after measurements have been made, and 3 new proteins are stained in their place. A robot carried out this task 18 times in total over the course of 18 days. Finally, a computer combines all of the individual photos into a single microscope image that shows 53 distinct proteins. These proteins offer details on the operation of the many retinal cell types, including the rods, cones, and ganglion cells. The researchers added information on which genes are read by specific cells to this visual data of retinal proteins.

Gain insights into retinitis pigmentosa

In order to create a time series of pictures and genetic data that accurately depicts the whole 39-week growth of retinal organoids, the researchers conducted all these studies on organoids that were at different ages and at different stages of development. They posted their image data as well as other retinal development research on the publicly accessible EyeSee4is website. 

Until now, researchers have focused on understanding how a healthy retina develops, but they intend to purposely interfere with retinal organoid development in the future using medications or genetic alterations to learn more about conditions like retinitis pigmentosa. 

The goal of the study is to determine when this process starts and how to halt it. In order to construct an atlas that details the growth of human organoids and tissues, Treutlein and her colleagues are now working on adapting the new precise mapping approach to additional tissue types, such as various tumor tissues and various parts of the human brain.

The results have been published in the journal Nature.

Study Abstract: 

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.