Project develops new stem cell lines whose receptors can be activated by blue light

A research project led by the IMC University of Applied Sciences Krems has been successfully completed and provides an excellent basis for further projects. The team led by Prof. Christoph Wiesner from the Institute of Biotechnology has succeeded in genetically modifying special receptors (Toll-like receptors, TLRs) on stem cells so that they can be activated by blue light.

Such “optogenetic” techniques could be used to precisely control biological signaling pathways in cells, validate them under physiologically relevant conditions, and generate disease models. These new optogenetic cell lines will also make a valuable contribution to understanding disease mechanisms and developing innovative, targeted therapeutic approaches.

The group led by Wiesner, holder of the research professorship “Cellomics / High Content Screening” at the IMC Krems, has been working on optogenetics for several years – a young research field that deals with the targeted control of cells using light.

The aim of the now successfully completed project was to develop new stem cell lines (MSCs, mesenchymal stromal cells) whose receptors were genetically modified by the incorporation of light-sensitive proteins so that they can be activated by blue light.

The work appears in the journal Limits of immunology And International Journal of Molecular Sciences.

MSCs are all-rounders

“In our project,” explains Prof. Wiesner, “we worked with so-called MSCs, or mesenchymal stromal cells. These are adult stem cells that occur in different tissues and can differentiate into different cell types.”

MSCs exist in the body in two different states (MSC1 and MSC2), which have different functions: The MSC1 cells have a pro-inflammatory effect, i.e. they promote inflammatory reactions and thus support the immune system in fighting infections and tumors.

The anti-inflammatory MSC2 cells, on the other hand, dampen inflammatory reactions in the body and are therefore useful in chronic inflammation, autoimmune diseases or to promote tissue repair after injuries. It is known that all MSCs carry special receptors – TLRs – on their cell surface, which recognize the molecular pattern of pathogens when they come into contact with them and trigger an immune response via subsequent signaling pathways.

However, the exact mechanisms by which the activation of different TLRs leads to the development of the two MSC forms are still poorly understood – a fact that Prof. Wiesner’s research team has addressed.

A question of regulation

Based on the hypothesis that MSCs can perform different functions depending on the activated TLR type and the strength of the stimulus (e.g. pro-inflammatory and anti-inflammatory, antibacterial or regenerative tasks), transgenic and optogenetic approaches should help to elucidate the mechanisms that lead to the polarization of MSCs into the two forms MSC1 and MSC2.

“To do this, we incorporated light-sensitive proteins into the TLRs so that we could switch the receptors on with light and off again with darkness,” explains Prof. Wiesner.

In particular, it was shown that TLR4 and TLR10 could be easily controlled by light after incorporation into the cell lines. The following observations proved that the optogenetic constructs worked perfectly: The activation of TLR4 led to the production of pro-inflammatory molecules, similar to a bacterial infection, while the activation of TLR10 regulated both pro-inflammatory and anti-inflammatory molecules.

A comprehensive analysis of the supernatant of the cultured MSC cell lines revealed numerous proteins that indicate the regenerative potential of the cells and the accelerated bone cell formation after TLR10 activation. This makes the new cell lines useful tools for studying the mechanisms of TLR4 and TLR10 activation and could provide new approaches for therapeutic strategies.

The ESPRIT project with Anna Stierschneider, senior postdoc in Christoph Wiesner’s research group, shows that the new optogenetic cell lines can be tested not only in individual experiments, in which miniaturized (0.2–0.5 mm in size), physiologically relevant 3D heterotypic cell models are established in vitro.

The optogenetic stem cells are integrated into heterotypic tumor cells (colorectal adenocarcinoma); 96 of these miniaturized tumors are cultured in parallel and the optogenetic approach is tested for its anticarcinogenic potential. Initial experiments are promising.

The successfully completed project not only resulted in these published studies, but also in further research projects and valuable collaborations – including the follow-up project ESPRIT and a training program for doctoral students (doc.funds), which the IMC Krems coordinates together with the FH Krems and the MedUni Vienna.

Together with ABS Biotechnology GmbH, a GFF application was submitted to introduce optogenetic constructs into inducible pluripotent cells and to investigate their effects on cardiac myocytes and macrophages.

In collaboration with KL Krems and UPEC University (Paris), the effect of TLR-activated mesenchymal stem cell supernatants on neurons and in Alzheimer’s disease models is being investigated.