Poster N15 - An in vitro Gut-Brain Axis Model for Studying the Role of Enteroendocrine Pathway

Introduction: Dysregulation of the gut-brain axis (GBA) is implicated in a spectrum of pathologies ranging from inflammatory bowel disease to neurodegenerative diseases. Untangling the causality of multifaceted diseases involving the GBA presents challenges in animal models, given the intricate systemic interactions compounded by interspecies variations in metabolism and immunology. This underscores the necessity for causality-driven, human-derived, preclinical GBA models that embrace an engineering-oriented conceptualization of diseases to capture the critical yet intricate physiological phenomena in vitro in a scalable manner.
Yet how the signals from gut reach the brain is still debated, the GBA has distinct pathways based on (i) vagus or enteric nervous pathway transduced to the central nervous system, (ii) endocrine, (iii) neuroimmune, (iv) humoral-mediated systemic circulation. Of particular interest are enteroendocrine cells (EECs), specialized intestinal epithelial cells responsible for producing hormones that convey information regarding nutrients, pain, and microbiome-related data to the brain and other organs. Recent investigations have unveiled a broader spectrum of hormone profiles and communication modalities associated with EECs, including direct synaptic communication with peripheral neurons.
Current EEC models present inherent limitations, including rare EECs population in organoids and the lack of physiological diversity in cell lines. In this context, we propose the development of a biofabricated in vitro EEC model as a platform for investigating the role of EECs in the GBA. We postulate that providing EECs with gut-specific biochemical and biophysical cues will induce epigenetic reprogramming of cells into diverse composition, thereby yielding a more faithful representation of EEC function.

Materials and

Methods: To examine the influence of gut-specific biochemical and biophysical signals on EECs behavior, we developed a gut-specific biomaterial, colon-derived decellularized extracellular matrix (colon dECM). We employed the NCI-H716 EEC cell line to examine the effects of diverse biochemical cues within a two-dimensional (2D) context. Specifically, NCI-H716 cells were cultured on surfaces coated with either non-coated, Matrigel-coated, or colon dECM. Under the influence of different ECM mateirals, we assessed alterations in cellular morphology, gene expression associated with epigenetic modifications influencing cellular fate, and responsiveness to microbial metabolites such as short-chain fatty acids (SCFAs).
Subsequently, we transitioned to a 3D environment with different tissue geometries. We implemented a 3D bioprinting technique to mimic the anatomical configuration of the native gut and compared with conventional dome shape. We then progressed to a three-dimensional (3D) milieu featuring diverse tissue architectures. Employing 3D bioprinting, we replicated the anatomical structure of the native gut and compared it with the conventional dome-shaped configuration. Utilizing the 3D tissue constructs, we evaluated the expression levels of genes involved in the maturation of EECs, neurotransmitter synthesis, and augmentation of neurotransmitter production mediated by SCFAs. Additionally, we investigated alterations in EEC functionality of the tissues under inflammatory conditions, simulating the phenomenon of leaky gut.
Finally, we harvested the secretome from the intestinal tissues and administered it to human neural progenitor cells, specifically ReNcell, possessing the capability to differentiate into both neurons and glial cells. Then we assessed the expression of genes important for the maturation and function of neurons and microglia.

Results, Conclusions, and Discussions: The biochemical cues derived from the colon dECM gave distinct morphologies to the NCI-H716 cells compared to Matrigel. In the Matrigel-coated group, cells exhibited morphological features consistent with the typical differentiation patterns observed in adherent cultures of NCI cells. In contrast, the dECM-coated group displayed a broader spectrum of morphological alterations, including the presence of granule-like structures, notable increases in cell body size, or a combination thereof.
To elucidate the underlying cause of these morphological variations, we examined the expression of various phenotype-related genes in the gut epithelium. In comparison to the control, both ECM-coated groups demonstrated dynamic transcriptional changes, indicative of heightened cellular plasticity. Subsequently, to ascertain whether these transcriptional adjustments correlated with augmented enteroendocrine functionality, we quantified serotonin levels in the ECM-coated groups. However, no discernible discrepancies were discerned among the groups. Interestingly, transitioning to the 3D environment, particularly the tubular geometry, markedly enhanced protein expression levels and in vivo-like localization. A pronounced localization of mucin expression in the luminal direction in the tube group was observed. In addition, the 3D tubular gut tissues showed significanlty increased synthesis of various neurotransmitters, such as serotonin and histamine, comparable to known clinical values. Inducing inflammation to the tubular gut tissue showed physiologically relevant alterations in the EEC cellular composition and function. These findings demonstrates synergistic impact of gut-specific biochemical and biophysical signals in augmenting EEC function in vitro.
Finally, the secretome from the healthy gut tissues increased the differentation of the neural progenitor cells into mature neurons and microglia, while the secretome from the leaky gut tissues triggered increase of proinflammatory cytokines. Collectively, our model showed promising potential in investigating the involvement of EECs in the GBA in vitro.

Acknowledgements (Optional): This research was supported by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (21A0104L1, Republic of Korea).