They look like thunderclouds in pinhead format: organoids. The three-dimensional cell cultures play an important role in medical and clinical research because they reproduce tissue structures and organ functions in the petri dish. Scientists can use them to understand how diseases arise, organs develop or drugs work. With the help of single-cell technologies, they penetrate to the molecular level of the cells. Thanks to spatial transcriptomics, they can even be observed in 3D and which genes are active at which location in the organoid over time.
The "miniature organs" are mostly developed from stem cells. These are cells that are not yet or barely differentiated. They can develop into any cell type, such as heart or kidney cells, muscle cells or neurons. Scientists "feed" them with growth factors and embed them in a nutrient solution. There they clump together into tiny cell clumps in which they finally function and interact as if they were in real tissue. Until now, it has hardly been possible to control this process. Researchers led by Professor Nikolaus Rajewsky, Director of the Berlin Institute for Medical Systems Biology at the Max Delbrück Center (MDC-BIMSB), now describe in the scientific journal Nature Methods a technology with which they can trigger it, control it and observe it in spatial and temporal resolution. "To do this, we have combined spatial transcriptomics with optogenetics," says first author Dr. Ivano Legnini. "This enables us not only to control gene expression in living cells, but also to observe its course."
Light sensors activate or block genes
In optogenetics, natural or artificially produced "light sensors" are inserted into cells. When light falls on the sensors, they activate or block the genes in the cells - depending on how they are programmed. Legnini incorporated such light sensors into neuron precursor cells developed from stem cells to form neural organoids. To do this, he worked with the team from the Organoids Technology Platform led by Dr Agnieszka Rybak-Wolf and the Systems Biology of Neuronal Cell and Tissue Differentiation group led by Dr Robert Patrick Zinzen. The researchers wanted to understand how the nervous system develops in the human embryo. Morphogens play a key role in this - molecules that signal to the neuronal precursor cells whether they should become neurons in the front part of the brain or in the back part of the spinal cord, for example. The combination of these molecules generates typical patterns of gene expression during development.
With the help of light, the researchers activated one of these morphogens, namely Sonic Hedgehog (SHH). The subsequent spatially resolved single-cell analyses showed that the cells then arranged themselves into typically patterned organoids. The researchers generated the light pulse in two ways: either with the help of a laser microscope or with a digital micromirror microscope, which Rajewsky's group developed together with Dr. Andrew Woehler. At the time, Andrew Woehler was head of the Max Delbrück Center's light microscopy platform; since November 2022, he has headed the Experimental Technologies Department at the Howard Hughes Medical Institute in Ashburn, USA. A chip with several hundred thousand tiny mirrors is inserted into this special microscope. These can be programmed so that the microscope can create complex patterns of illumination on a sample - unlike with a laser, which only hits a single spot at a time.
Precise - with potential for improvement
"With our method, we can reproduce processes related to gene expression in tissues very precisely in the Petri dish," says Ivano Legnini. Since March of this year, he has been setting up his own research group at the Human Technopole in Milan. There, among other things, he wants to improve the spatial and temporal resolution of the technology and make it applicable to other organoids. Nikolaus Rajewsky also wants to continue refining the method: "I am very much looking forward to working with optogenetics experts to further improve the technology and apply it to clinically relevant human organoid models."
Original publication: Spatiotemporal, optogenetic control of gene expression in organoids (Legnini et al., 2023)
Scientific contact: Prof. Nikolaus Rajewsky (rajewsky@mdc-berlin.de), AG Systems Biology of Gene Regulatory Elements, Berlin Institute of Medical Systems Biology of the Max Delbrück Center (MDC-BIMSB), Berlin
Source: idw press release (in German only)