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When the “second brain” fails – therapeutic options from the field of regenerative medicine

Researchers from the Center for Regenerative Biology and Medicine ZRM in Tübingen are focused on the enteric nervous system of the gastrointestinal tract with the aim of developing cell-based therapies for the treatment of bowel diseases. The researchers are working with local partners in a BMBF-funded project that is seeking to develop therapies for Hirschsprung’s disease.

PD Dr. Lothar Just is investigating the enteric nervous system in the gastrointestinal tract and developing concepts for the treatment of “second brain” diseases. © Lothar Just, University of Tübingen

The enteric nervous system of the human gastrointestinal tract, also known as “second brain” or gut brain”, consists of some one hundred million neurons. The enteric nervous system is capabable of autonomous functions and together with the parasympathetic and sympathetic nervous systems constitutes the autonomic nervous system. Despite its dimensions, the “second brain” has not yet been a major focus of neurological research. “Little is still known about the enteric nervous system. But it plays a much bigger role in the human body than previously assumed. Amongst other things, studies have highlighted the link between the enteric nervous system and the immune system and there is also evidence that the enteric nervous system has a modulatory effect on the immune system,” said PD Dr. Lothar Just from the Center for Regenerative Biology and Medicine ZRM at the University of Tübingen and the University Hospital of Tübingen. Just has been focusing on the “second brain” for a number of years now and tells us that there is growing evidence to show that there is an interaction between the enteric and central nervous systems. “According to current knowledge, around 90 per cent of all enteric neurons are linked in some way with the central nervous system. Therefore it does not come as a surprise that the enteric system has a decisive influence on our mood and health.

Diseases of the enteric nervous system can have severe and life-threatening consequences. Lothar Just is coordinating a three-year BMBF project, which was granted funding in 2009, focusing on developing therapies for the treatment of Hirschsprung’s disease, a genetic disorder caused by the partial or complete lack of enteric neurons in certain intestinal regions, particularly in the colon. The disease causes disorders in intestinal movements and hence in stool transit. The symptoms can range from severe constipation to life-threatening bowel obstruction and gastrointestinal perforation. The disease affects on average one in 5,000 newborn babies. Although it is possible to alleviate disease symptoms by surgically removing affected colon segments, many patients still suffer chronic problems after surgery.

The goal: regeneration of missing nerve cells

As part of the BMBF project, the researchers are looking for a cell-based therapy that can be used to regenerate neurons in the affected colon segment. Scientific studies undertaken by Tübingen researchers have shown that the intestines of children and adults contain immature neural precursor cells, which can be expanded in cell culture and differentiated into functional neurons of the enteric system. These findings form the basis for the development of a regenerative therapy for Hirschsprung’s disease. Over the last three years, the cooperative project has made considerable progress. “The results are so promising that we will continue with our project when BMBF funding comes to an end. We are applying for further funding and hope that the BMBF will provide us with funds until early next year. In addition, we are also going to develop follow-up projects aimed at taking the therapy to clinical application,” said Just.

The cooperative project also involves researchers and doctors from the Department of Paediatric Surgery and Paediatric Urology at the University Children's Hospital in Tübingen (UKT), the NMI Natural and Medical Sciences Institute in Reutlingen and Weinheim-based Naturin Viscofan GmbH. Since it began in 2009, the project has made considerable progress and enabled the researchers to optimize the isolation and preparation of neurons from murine and human colon segments. “When we isolate neurons, we first mechanically separate the outer muscle layer from the rest of the intestinal wall. The outer muscle layer contains a relatively large number of enteric neurons. Once this is done, we use enzymes to further process the cells,” said Just.

How neural precursor cells are isolated, expanded and differentiated in the laboratory

Immunofluorescence image of the enteric system in a mouse. The neurons form a complex network in the intestinal wall. © Lothar Just, University of Tübingen

In addition to optimizing the procedure of isolating neurons, the researchers also needed to optimize the cell culture procedure, something they have since successfully done. They had to figure out the ingredients for the culture media required for growing the cells as well as finding biomaterial that was suited for use as a scaffold on which the cells could grow. That’s where the company Naturin Viscofan GmbH came in. Naturin Viscofan GmbH specializes in the use of animal collagens for a broad range of different applications, including in the food industry. The company tested different types of collagen to find out which were the most suited for the cell culture experiments being carried out by the Tübingen researchers. The collagen also underwent different types of treatment. “There are components that make cells grow in a specific orientation and others that make the cellular protrusions grow in any direction whatsoever. We do not yet understand how this is achieved on the molecular level, but we have found a material that is excellently suited for finding out more. We use a membrane that can be seeded on both sides with cells and that promotes their propagation and differentiation,” said Just.

The researchers led by Dr. Florian Obermayr at the Department of Paediatric Surgery at the University Children's Hospital of Tübingen have implanted these cells into the intestines of healthy mice. “The cells survived in the mice and we were also able to follow their behaviour. This success is a major prerequisite for our future work,” said Just. One of the project’s greatest challenges was to prove that the differentiated cells were neurons. Prof. Dr. Elke Günther from the NMI in Reulingen was responsible for providing the necessary evidence. “We had to develop a staining method involving multiple dyes in order to show two things: first, that the cells divided and second, that the electrophysiologically patched cells were indeed the ones we were looking for, i.e. differentiated cells with sodium channels,” explained Just. Transmembrane voltage-gated sodium channels are characteristic of neurons. They directionally propagate electrical signals, which can be triggered and measured with electrophysiogical methods.

Clinical application is tested in animal models

This is what the researchers have achieved so far: the team has obtained functionally active neurons containing voltage-gated sodium channels. This means that the precursor cells isolated from the intestines have developed into functional neurons after implantation and survived at least in the animal model. Further research will need to concentrate on the sub-classification of the precursor cells at the time of isolation in order to narrow down the number of cells that need to be tested. The researchers are well on their way; they have already identified interesting biomarker candidates with the potential to differentiate neural precursor cells from other cells.

Based on these findings, the researchers will now establish animal models involving larger rodents and pigs with the aim of characterizing the improvements that have resulted from the transplanted cells. This also means that they will have to differentiate between excitatory and inhibitory neurons. Just explains: “The colon contains excitatory and inhibitory neurons; functional investigations and therapies need to target either one. This is why we need to be able to differentiate between these two types of neurons.” Just also has plans to involve human geneticists in future projects in order to be able to analyze genetic influences on the enteric nervous system.

Further information:

PD Dr. Lothar Just
University of Tübingen
Institute of Anatomy
Österbergstr. 3
72074 Tübingen
Tel.: +49 (0)7071/ 29 - 72 176
E-mail: ljust(at)anatom.uni-tuebingen.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/when-the-second-brain-fails-therapeutic-options-from-the-field-of-regenerative-medicine