Abstract

Insights into Interactions of the Microbiome-gut-brain Axis on a Microfluidic Chip

The gut-brain axis (GBA) is a bi-directional communication system that connects the central nervous system and the gastrointestinal system. Interaction can involve immune, endocrine or metabolic pathways, or the vagus nerve as the most direct route. The functioning of the GBA is also considerably impacted by the bacteria in the intestine – the gut microbiome. Dysregulation is implicated in a large number of neurodegenerative diseases and conditions [1]. For an improved fundamental understanding of the microbiome-gut-brain axis and its functioning in health and disease, we are translating the organ system to a custom microfluidic chip using pluripotent stem cells (PSCs).To establish a working co-culture comprised of a microbiome, an intestinal epithelium connected to a vagus nerve and brain tissue, the components of the GBA were recapitulated using human pluripotent stem cells. Initially, human intestinal organoids (HIOs) were generated from PSCs by exposure to growth factors inspired by embryonic intestinal development as described in established protocols [2]. The resulting mid/hindgut tube spheroids stained positive for transcription factors SOX17, FOXA2 and CDX2 - markers typical for differentiation towards intestinal tissue [3]. These spheroids were then expanded in Matrigel droplets giving rise to three-dimensional intestinal tissue consisting of a polarized, columnar epithelium. The tissue also featured relevant cellular subtypes of the intestinal epithelium such as enterocytes, goblet cells, Paneth cells and enteroendocrine cells. We also introduced small intestine-specific bacterial strains such as Lactobacillus rhamnosus to the intestinal compartment.To obtain a vagus nerve, PSCs were differentiated into human vagal neural crest cells (NCCs) as a precursor. Using GDNF, the vagal NCCs were further defined to ultimately form enteric neurons characterized by the expression of neuronal markers such as TUBB3 and MAP2, and the enteric neuron marker PHOX2B [4]. To model the cerebral component, PSCs were differentiated into neuroepithelial (NE) tissue using dual SMAD inhibitors and Wnt blocker. The NE was further differentiated into hippocampal organoids using a modified version of a previously reported method [5]. In addition to neuronal markers, the organoids tested positive for regional hippocampal markers Prox1, KA1, and Zbtb20. Electrophysiology was observed using Calcium imaging, FluoVolt, and multielectrode arrays (MEAs).  Spatially arranging intestinal organoids, vagal neurons and brain organoids on a microfluidic PDMS chip allowed us insight in their convergence and interactions. Ultimately, we will study the microbiome-gut-brain axis in connection with neurodegenerative disorders such as Parkinson’s disease or Alzheimer’s disease.