Faculty, Staff and Student Publications
Language
English
Publication Date
1-17-2025
Journal
JoVE: Journal of Visualized Experiments
DOI
10.3791/67702
PMID
39895623
PMCID
PMC13102250
PubMedCentral® Posted Date
4-23-2026
PubMedCentral® Full Text Version
Author MSS
Abstract
The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing. Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into >9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.
Keywords
Humans, Organoids, Animals, Mice, Pluripotent Stem Cells, Intestine, Small, Coculture Techniques, Enteric Nervous System, Neural Crest
Published Open-Access
yes
Recommended Citation
Rachel C Bordelon, Madushani Herath, and Allison L Speer, "Innervation of Human Intestinal Organoids" (2025). Faculty, Staff and Student Publications. 4130.
https://digitalcommons.library.tmc.edu/uthmed_docs/4130