What types of cells are found in what human tissue and where? What genes are active in individual cells and what proteins are found there? The answers to these questions and more will be provided by a specialized atlas, in particular how different tissues are formed during embryonic development and what causes diseases. By creating this atlas, the researchers aim to map not only tissue isolated directly from humans, but also structures called organoids. These are three-dimensional clumps of tissue that are grown in the lab and grow in a similar way to human organs, but on a small scale.
“The advantage of organoids is that we can intervene in their development and test active substances on them, which allows us to learn more about healthy tissue and disease,” explains Barbara Treutlein, Professor of Quantitative Developmental Biology in the Department of Developmental Sciences. of Biosystems. and Engineering at ETH Zurich in Basel.
To help produce such an atlas, Treutlein, together with researchers from the Universities of Zurich and Basel, has developed an approach to collect and collate a wealth of information on organoids and their development. The research team applied this approach to human retinal organoids, which were derived from stem cells.
Many proteins visible simultaneously
At the heart of the methods the scientists used for their approach was the 4i technology: iterative indirect immunofluorescence imaging. This new imaging technique can visualize several dozen proteins in a thin section of tissue at high resolution using fluorescence microscopy. The 4i technology was developed a few years ago by Lucas Pelkmans, a professor at the University of Zurich and co-author of the study that has just been published in the scientific journal Nature Biotechnology. It is in this study that the researchers applied this method to organoids for the first time.
Typically, researchers use fluorescence microscopy to highlight three proteins in a tissue, each with a different fluorescent dye. For technical reasons, it is not possible to stain more than five proteins at a time. In the 4i technology, three dyes are used, but these are washed out of the tissue sample after the measurements have been taken and three new proteins are stained. This step was performed 18 times, by a robot, and the process took a total of 18 days. Finally, a computer merges the individual images into a single microscopy image showing 53 different proteins. They provide information about the function of the individual cell types that make up the retina; for example, rods, cones, and ganglion cells.
The researchers have supplemented this visual information from retinal proteins with information about which genes are read in individual cells.
High spatial and temporal resolution
The scientists performed all of these analyzes on organoids of different ages and therefore at different stages of development. In this way, they were able to create a time series of images and genetic information describing the entire 39-week development of retinal organoids. “We can use this time series to show how organoid tissue slowly accumulates, where and when what types of cells proliferate, and where synapses are located. The processes are comparable to those in the formation of the retina during embryonic development.” says Gray Camp, a professor at the University of Basel and lead author of this study.
The researchers published their image information and further findings on retinal development on a publicly accessible website: EyeSee4is.
Other types of fabrics planned
Until now, scientists have been studying how a healthy retina develops, but in the future they hope to deliberately disrupt the development of retinal organoids with drugs or genetic modification. “This will give us new insights into diseases such as retinitis pigmentosa, an inherited condition that causes the light-sensitive receptors in the retina to gradually degenerate, ultimately leading to blindness,” says Camp. Researchers want to know when this process starts and how it can be stopped.
Treutlein and his colleagues are also working on applying the new detailed mapping approach to other tissue types, such as different sections of the human brain and various tumor tissues. Step by step, this will create an atlas that provides information on the development of human organoids and tissues.
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