Objective

The gastrointestinal tract is a vital and complex organ bearing multiple functions in humans. The first of these is nutrition, supported by the intestinal epithelial barrier and the enteric nervous system (ENS), which regulates both gut motility and secretions. During embryogenesis, the co-development of ENS progenitor cells and embryonic germ layers is crucial for the establishment and maturation of different regions of the digestive tract, particularly the small intestine and colon. However, the crosstalk and trophic mechanisms by which the ENS contributes to gut development remain poorly understood.

Methods

To explore the influence of the ENS during embryogenesis, we developed human intestinal (HIO) and colonic (HCO) organoid models derived from human-induced pluripotent stem cells (hiPSCs), co-cultured with vagal neural crest cells (vNCCs) for four months. An exploratory bioinformatics analysis was performed using bulk RNA sequencing data from these organoids (HIO ± vNCCs ; HCO ± vNCCs) across 16 time points. We first conducted a Weighted Gene Co-expression Network Analysis (WGCNA) in combination with time-course analysis using the MaSigPro tool, and we then performed a gene ontology analysis to identify upregulated pathways and biological processes potentially mediated by the ENS.

Results

This work revealed distinct gene expression patterns depending on the presence or absence of vNCCs in the organoids, with specific gene modules whose expression was either positively or negatively correlated with the presence of ENS progenitors. Several time-specific gene modules also emerged, showing expression patterns restricted to early or late stages of in vitro cultures. In small intestinal organoids, these modules were enriched in genes related to muscular and vascular signatures, including ontologies related to mesodermal development and angiogenesis, supporting a trophic role of the ENS on the mesenchymal compartment during human gut development. Furthermore, distinct gene modules specific to colonic organoids, particularly one associated with immune responses, highlight regional differences in ENS-mediated processes and suggest segment-specific effects in gut formation.

Conclusion

In conclusion, this study provides new insights into the regulatory mechanisms orchestrated by the ENS during gut development and opens avenues for further research into neuropathic disorders such as Hirschsprung’s disease.