Towards a microbiome-gut-brain axis on a chip - Insights into multi-organ interactions underlying gut-brain communication
Lena Koch is a PhD student in the Department of Applied Stem Cell Technologies. Promotors are dr. K. Broersen and prof.dr. P.C.J.J. Passier from the Faculty of Science & Technology.
The gut, its microbiota and the brain communicate with each other via the microbiome-gut-brain axis (MGBA) which represents the bi-directional path for interaction between the gastrointestinal tract and the central nervous system. The vagus nerve offers a direct route of communication within the MGBA as many modulatory effects of gut microbiota are directly facilitated via vagal activation. Dysregulation of the relationship between the microbiota and the host is implicated in a large number of diseases and pathological conditions involving the MGBA. Increasing evidence demonstrated that, specifically, neurodegenerative diseases (NDs) are not only confined to the brain but are progressively viewed in the context of alterations in the intestinal physiology. However, the exact mechanisms underlying remain unclear. While current in vivo models lack transferability of results and raise ethical issues, in vitro models to study gut-brain interactions often have unilateral approaches and lack representativeness of the complex multi-organ system. This thesis describes our efforts into recreating various components of the microbiome-gut-brain axis as well as creating a custom microfluidic chip to enable parallel multi-organ culture. Our aim is to gain a deeper understanding of processes underlying gut-brain communication.
In chapter 2 we give an introduction to organ-on-chip technology which combines stem cell-derived miniature organs with microfluidic platforms to overcome challenges of conventional well plate-based culture methods, and help study fundamental processes and contribute to understanding disease pathology. We provide an overview of key achievements three dimensional organoids recapitulating organs such as brain, retina, spinal cord, liver and kidney and discuss how integrating organoids into microfluidic systems has addressed some of these issues, thereby enhancing viability, functionality and physiological relevance of these models. Recognizing the importance of organ-organ interactions in regulating organ functionality, we explore the latest advancements and limitations in multi-organ models. We then explore major challenges in creating representative and reproducible organ-on-chip models and give future directions. Finally, we explore the possible applications of organoid-on-chip systems in both in vivo and in vitro studies with an outlook on medicine, disease modeling and regenerative medicine.
In chapter 3 we focus on the brain component of the microbiome-gut-brain axis and present the development of cerebral organoids from pluripotent stem cells. We created a cost-effective and efficient method to optimize a crucial first step in the process of organoid differentiation. This optimization reduced size variability and improved the overall yield of successfully formed embryoid bodies (EBs) across a range of seeding densities. The resulting homogenous EBs ensured more consistent development of organoids thereby contributing to higher reproducibility of future studies. Chapter 4 continues on cerebral organoids, and presents a visual step-by-step guide for an optimized protocol for generating, maintaining, maturing and characterizing human iPSC-derived cerebral organoids. We addressed specific issues related to organoid maturation, necrosis, variability, and batch effects. This protocol was designed to enable long-term culture of organoids allowing the identification of physiologic and pathogenic mechanisms underlying human brain aging.
In chapter 5, we shifted our focus from the brain to the gut and present a comprehensive overview of the regulation of physiological mucus layer development in the human intestine. Dysregulation of the mucin layer is implicated in a range of pathologies implicating gut-brain interactions, especially inflammatory processes. We showed that the production and release of mucin-2 from intestinal goblet cells is regulated in a multi-factorial manner, often converging with inflammatory pathways. We further highlighted that physiological mucus layer development is highly dependent on parallel input form the commensal microbiome and the enteric nervous system and vagus nerve. Based on our findings, we offered a perspective on current in vitro models of the intestine that often lack a functional mucus layer and suggest innovative strategies to drive a more accurate recapitulation of intestinal function. In chapter 6, we focus on a specific pathway in the mucin-2 production where we identified a lack sufficient research and evidence for in chapter 5. Using an in vitro model of mucin-producing goblet cells, HT29-mtx cells, we elucidated the molecular trafficking mechanism of mucin-2 between the endoplasmic reticulum (ER) and the Golgi apparatus. We discovered a novel molecular pathway involving COPII-coated vesicles, coordinated by proteins TANGO1, cTAGE5, and KLHL12, with KLHL12 playing a critical role in enlarging vesicles to accommodate the large size of mucin-2, similar to the pathway reported for procollagen I. Additionally, we found that TANGO1 expression is regulated by transforming growth factor-beta (TGF-β), which is often abnormal in IBD. Our findings indicated that disruption of the COPII-mediated mucin-2 transport contributed to ER stress observed during inflammatory bowel disease, a pathology marked by a deficient mucus layer. Gene expression analysis of colonic tissues from the 1000IBD project confirmed our findings showing elevated ER stress markers and dysregulation of TGF-β and mucin-2 expression during IBD. In chapter 7, we aimed to take first steps into modeling the intestinal epithelia and integrate microbiota to gain first insights into the development of this components for a complete model. We used an established immortalized cell line-based model of the intestine consisting of Caco-2 and HT29-mtx cells, representing enterocytes and mucin-producing goblet cells, respectively, to generate an epithelium featuring a functional mucus layer to facilitate microbiota integration. The probiotic bacterium Lactobacillus acidophilus (LA), was successfully introduced showing physiological interactions with the intestine and mucus layer. The presence of LA resulted in elevated mucin-2 expression in HT29-mtx cells. Barrier integrity measurements confirmed a commensal role of LA.
In chapter 8, we focused on taking first steps towards developing a microbiota-gut-brain axis model and present the design and fabrication of a microfluidic platform to support integration of this complex multi-organ system. We established differentiations for stem cell-derived hippocampus brain organoids (HBOs) and vagus-nerve like neurons (VNs) and subsequently characterized them for the expression of relevant markers and functionality. As a first step towards establishing the MGBA model, we integrated HBOs and VNs in a co-culture on the microfluidic chip and examined their interactions. Our study demonstrated that the chip supports their growth and proliferation in their designated chip compartments, while specialized microchannels facilitated axonal extensions into either the HBO or VN compartment leading to reciprocal innervation of both tissues. Preliminary studies indicated the physiological functionality of this connection as beneficial effects from microbiota-derived metabolites were mediated to the HBOs by the presence of the VN.