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Identification and characterization of enteric nervous system stem cells

Periodic Reporting for period 1 - IDENSTEM (Identification and characterization of enteric nervous system stem cells)

Reporting period: 2020-01-01 to 2021-12-31

Why is important for society?

The enteric nervous system (ENS) represents the largest collection of neurons and glial cells outside the central nervous system (CNS) which are intrinsic to the gastrointestinal tract and are essential for digestive function. The ENS controls virtually every aspect of gastrointestinal physiology including intestinal motility, epithelial secretion, and blood flow, while recent evidence indicates that enteric neurons and glia contribute to intestinal epithelial barrier functions and immune homeostasis. Not surprisingly, ENS activity also impacts the dynamics and composition of the complex consortia of microorganisms that colonise the gut shortly after birth. While the ENS regulates gastrointestinal physiology independently of CNS input, it is likely to serve as a relay station along the bidirectional gut-brain axis communication pathways. The critical contributions of the ENS to gastrointestinal physiology and homeostasis are highlighted by life-threatening developmental disorders, such as Hirschsprung disease, characterised by failure of development of enteric ganglia in the distal colon.

What is the problem?

The identity and properties of ENS neural stem cells (ENSCs) remain undefined and the cellular and molecular mechanisms that maintain ENS homeostasis in the face of adverse environmental influences or repair its neural circuitry following injury or disease, are unknown. We postulate that in vertebrates the EGC compartment exhibits NSC activity and aim to describe its cellular basis and regulation. Preliminary data suggest that a subpopulation of enteric glial cells expressing the Notch signaling target Hes5, undergo low rate proliferation and exhibit neurogenic potential.

What are the overall objectives?

Here, we aim to identify and characterize the cellular and molecular properties of the ENSSCs and to understand their contribution in maintaining ENS integrity under normal conditions or in response to injury.
In order to characterize these cells, we took advantage of a Hes5-CreERT2.R26tdTomato reporter mouse line to carry out inducible genetic lineage tracing of Hes5+ cells in the ENS. Administration of a single dose of tamoxifen (TM) to adult Hes5-CreERT2.R26tdTomato animals resulted in appearance shortly afterward (3 days) of tdTomato+ intra-ganglionic glial cells also known as type I glial cells. The Hes5+ cells are part of the Enteric Glial Cell (EGC) population as they co-express the neural crest (NC) lineage marker Sox10 and the canonical glial cell marker S100b. Following a 4-week chase was observed an increase in the average size of the tdTomato+ cell clusters, which also included cells expressing the pan-neuronal marker HuC/D. These findings suggested that under physiological conditions the ganglia of the mammalian myenteric plexus include a cell population that undergoes proliferation at a low rate and is capable of generating enteric neurons. Nevertheless, same experiments shown highly variability in the cluster size between time points whereas neuronal-like tdTomato+ numbers appeared to be consistently higher over time.

Following these set of experiments these animals were treated with EdU in the drinking water in order to address proliferation. EdU is a thymidine analogue that is incorporated in the cells while they are synthetizing their DNA. Surprisingly, a low number of Hes5+ cells were EdU positive meaning that not all of them were undergoing cell division. These results showed a low proliferative capacity of the entire Sox10+ glial cells not only restricted to the Hes5+ population. Furthermore, we failed to find any EdU+ tdTomato+ neuron.

To test proliferation under injury conditions, mice were treated with Dextran Sodium Sulfate (DSS) to induce colitis. DSS is a chemical agent that induces intestinal inflammation by damaging the epithelial layer mostly of the large intestine allowing the dissemination of bacteria into other tissues. Colon tissues were isolated after approximately 3 weeks of DSS treatment. After exhaustive counting of Sox10+ S100+ EdU+ glial cells, we can conclude that there are no significative differences in terms of cell proliferation after inflammation.

To address the importance of Notch signaling pathway in glial cell proliferation and neuronal regeneration our next aim was to study the behavior of the ENS in the absence of Notch signaling. As hes5 is a target of Notch we deleted RBPJk (a key downstream component of the canonical Notch signaling pathway) specifically in the Sox10+ glial cells. For this purpose, we generated the following mouse line: Sox10ERT2.tdTomato x RBPjk flox. In line with previous experiments, EdU was administrated in the drinking water, however, no differences in terms of proliferation or neuronal generation were observed compared with their littermate controls. All analysis was done using confocal imaging.

We were able to identify Hes5 gene expression patterns in existing data from the lab. Our work was completely disrupted in terms of animals and The Francis Crick facilities were not available during the pandemic. All of these together generated a delay in the implementation of IDENSTEM as it was originally planned. All necessary approvals in terms of animal experimentation were obtained, however, meeting all deadlines was not possible.
IDENSTEM increased my skills significantly. I have acquired new technical and managing skills that I didn’t have in the beginning. In terms of technical skills, I have got an entirely new spectrum of approaches to analyse the gut enteric nervous system. I have contributed to the lab with my previous immunology expertise adding experience in flow cytometry techniques and immunophenotyping. All of these together allowed me to think independently about my own experiments. Furthermore, I will be using the training budget from this MSCA fellowship to gain more managing skills by attending 2 different EMBO leadership courses that will give me more in-depth knowledge on how to become an independent investigator.

The Francis Crick Institute was the perfect environment to carry out my work having all the possibilities in terms of facilities and researchers. The communication between labs was great giving me the opportunity to have interesting discussions with other research groups. I think the timing was not perfect, due to the pandemic everything was much more difficult in terms of deadlines. I wish I could have had an extension to have the opportunity to take full advantage of IDENSTEM.
Enteric nervous system (Furness 2006)