research projects

Botulinum neurotoxins (BoNTs) are used to treat a range of neuromuscular dysfunctions and in aesthetic medicine as therapeutic agents. Each batch of pharmacological preparations of these highly potent neurotoxins, produced by Clostridium botulinum bacterial strains, must be tested for activity. Although alternatives exist, their activity is still determined to a large extent by the mouse lethality assay. Replacement methods approved to date are applicable only to single compounds, where highly specific neoepitope antibodies are used to detect the proteolytic activity of the small subunit of BoNTs. For batch testing of BoNTs, 400,000 mice are still used annually within Europe alone (as of 2018). New products tested exclusively in animals are no longer accepted by the European Medicines Agency (EMA). More than 40 subtypes and mosaic variants are now known in addition to BoNT serotypes A-H. Pharmacological use of diverse subtypes and new products is likely in the future, as resistance to individual products may emerge. A serotype-independent, animal-free assay that can be used for activity testing of BoNT-containing pharmacological products across the board does not yet exist, but is urgently needed.

The MoNLight-BoNT assay (Motor Neuron Light-BoNT Assay) is based on motor neurons (MNs) differentiated from human induced pluripotent stem cells (iPSCs). The activity of the pharmacological BoNT products is inversely proportional to the release of a reporter enzyme (luciferase) from the neurotransmitter-containing vesicles of MNs. The assay is serotype-independent and would therefore be a suitable replacement method to fully replace the mouse lethality assay associated with batch testing (Replace in the context of the 3Rs).

Funding: The development of the MoNLight-BoNT assay was supported by the German Federal Ministry of Education and Research (BMBF) (FKZ 031L0132B, 4/2017-9/2020). Cooperation with the research group of Prof. Dr. Gerhard Püschel, University of Potsdam, Institute of Nutritional Science, Department of Biochemistry of Nutrition.

Contact: Prof. Bettina Seeger

Publication:

Schenke M, Schjeide BM, Püschel GP, Seeger B (2020) Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays. Toxins (Basel) 12(5) doi:10.3390/toxins12050276

Schenke M, Prause H-C, Bergforth W, Przykopanski A, Rummel A, Klawonn F, Seeger B (2021) Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1. Toxins (Basel) 13(8):585 doi:10.3390/toxins13080585

Chronic pain is a major health problem, affecting around 20 to 50 % of the world's population. Therapy methods are largely developed in behavioral tests with induced pain in rodents. No pain treatment is possible, which leads to severe stress for the animals. However, therapeutics developed in animals are not always effective in humans. Therefore, two in vitro models will be developed in human cells to quantify neuron-mediated chronic pain with the highest possible predictive power. To this end, stem cell-derived sensory neurons (1.) will be used to develop a luciferase-based exocytosis assay to make the increased release of the neuropeptides involved in the chronic pain response (substance P and calcitonin gene-related peptides) easily quantifiable. In addition, (2.) an innervated skin model will be developed to quantify neurite outgrowth and the expression of chronic pain-associated regulated genes of pain receptors and ion channels involved in pain transmission in vitro. Proof-of-concept for the use of the models in pharmacology and toxicology will be provided by inhibiting induced neuropeptide release, induced neurite outgrowth and induced gene expression with therapeutically effective substances or by triggering them via the addition of exogenous substances. In this way, molecular signaling pathways of chronic pain development can be modeled in vitro, directly in human cells, in order to avoid unnecessary animal testing and to develop effective and safe therapeutics for humans. Following the project, the skin model will be transferred to the modeling of atopic dermatitis by using induced pluripotent stem cells and primary cells from patients in order to develop specific in vitro disease models for the development of new therapies in cooperation with industrial partners. The project will also serve as a basis for further cross-area in vitro modeling, such as the simulation of chronic joint pain.

funded by the Stiftung set

Contact: Prof. Bettina Seeger and PhD student Kathrin Lämmerhirt-Simmons

The intestine plays a crucial role for many diseases transmissible from animals to humans (so-called zoonoses). While various cell culture-based -systems are available for the study of disease mechanisms in mice, rats, and humans, the study of such processes in farm animals (e.g., pigs, cattle, or poultry) still requires the use of primary cell or organ cultures taken directly from the animal, or even the use of animals.

For this reason, new approaches to develop -models to study underlying molecular mechanisms of infections in farm animals are of great interest, especially in light of the fact that farm animals are often carriers or vectors of zoonotic pathogens. Therefore, the effects of bacterial toxins in a coculture of human intestinal absorptive enterocytes and goblet cells have been compared with the same effects in 2D seeded porcine intestinal organoids in the Ussing chamber system to characterize the underlying cell systems.

Funding: Project A2 within the joint project R2N - Replace and Reduce in Lower Saxony, funded by the Ministry of Science and Culture of Lower Saxony (MWK) in cooperation with Prof. Dr. Gerhard Breves, TiHo, Institute of Physiology and Cell Biology

Contact: Prof. Bettina Seeger, Ph.D

Publication:

Seeger B (2020) Farm Animal-derived Models of the Intestinal Epithelium: Recent Advances and Future Applications of Intestinal Organoids. Alternatives to laboratory animals : ATLA:261192920974026 doi:10.1177/0261192920974026

Hoffmann P, Burmester M, Langeheine M, Brehm R, Empl MT, Seeger B, Breves G (2021a) Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells. PLoS One 16(10):e0257824 doi:10.1371/journal.pone.0257824

Hoffmann P, Schnepel N, Langeheine M, Künnemann K, Grassl GA, Brehm R, Seeger B, Mazzuoli-Weber G, Breves G (2021b) Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine. PLoS One 16(8):e0256143 doi:10.1371/journal.pone.0256143

In the master thesis of Elena Wiebe, intestinal organoids differentiated from human iPSCs were infected with Staph. aureus under normoxic and low oxygen conditions. Staph. aureus is involved in acute intestinal infections that can cause nausea, vomiting, abdominal pain, and diarrhea. In collaboration with the group of Prof. Maren von Köckritz-Blickwede, TiHo, Institute of Biochemistry and RIZ, we are investigating molecular mechanisms in the infection of intestinal epithelial cells in intestinal organoids by Staph. aureus under normoxic and hypoxic oxygen conditions.

Contact: Prof. Bettina Seeger, Ph.D.

The pig is an important model for the study of human gastrointestinal diseases, as there are great similarities between the two species. However, there are also species-specific differences in pathophysiological processes, such as Yersinia enterocolitica infection, which leads to gastroenteritis in humans, whereas pigs remain asymptomatic. Intestinal organoids have become important tools to study the underlying mechanisms in recent years. During long-term culture, the expression of proliferating intestinal stem cells in particular is upregulated to support organoid self-renewal and proliferation. To generate models to study molecular mechanisms during bacterial infections, the aim of this study is to enrich the expression of specialized intestinal epithelial cells, such as M cells, goblet cells and enteroendocrine cells in porcine intestinal organoids.

Funding: Supported by a doctoral fellowship from the Schaumann Foundation (01/2020-12/2021).

Contact: Prof. Bettina Seeger