P0974 - Integration of the Enteric Nervous System (ENS) within Human Intestinal Organoids (HIOs) alters epithelial differentiation

Introduction: Short bowel syndrome is a devastating disease affecting a growing number of children. Current management is inadequate. Tissue-engineered intestine may provide a solution; however, complex intestinal functions have not been validated in existing models. The development of human intestinal organoids (HIOs) has provided a novel model to generate intestinal tissue, but HIOs lack an enteric nervous system (ENS), which is critical for function. Prior studies have incorporated the ENS into HIOs using different protocols with variable success. We sought to optimize the integration of the ENS into HIOs by using a recent chemically defined protocol to better generate enteric neural crest cells (ENCCs). We hypothesized that the ENCCs would provide early developmental cues to drive HIO epithelial differentiation and vascular formation.

Methods: HIOs and ENCCs were generated from hESCs and GFP-labeled hESCs, respectively, using previously published protocols. HIOs±ENCCs were suspended in Matrigel and grown in vitro. Fluorescence microscopy was used to track ENCCs. HIOs±ENCCs were collected after 3 days of co-culture. Immunofluorescence (IF) staining and qRT-PCR were performed.

Results: ENCCs are SOX10 positive and demonstrate upregulated ENCC lineage marker PAX3 and vagal neural crest markers HOX3B and HOX5B. ENCCs demonstrate neuronal (TUJ1) and glial (GFAP, BLBP) proteins on IF. ENCCs successfully integrated into the HIOs on fluorescence microscopy. Interestingly, HIOs+ENCCs demonstrated decreased CHGA, MUC2, LYZ, SI, ATOH1, HES1, and PECAM on qRT-PCR (Figure 1).

Conclusions: ENCCs integrated with HIOs and provided developmental cues that altered HIO epithelial differentiation and vascular formation. Future studies will focus on long-term effects.