Innervation of Human Intestinal Organoids

Summary

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

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.

Introduction

The human small intestine is a complex, multilayered organ that carries out numerous essential functions such as digestion, nutrient absorption, fluid regulation, immune barrier function, and motility. Numerous clinical diseases, such as short bowel syndrome, enteropathies, or motility disorders, are characterized by critical reduction of intestinal mass or disruptions of normal physiology leading to significant morbidity and mortality^1,2,3,4. Current treatment options often include surgery to remove the dysfunctional intestine at the cost o....

Protocol

Human embryonic stem cell (hESC) line H9 was sourced from WiCell (Madison, WI) and all experiments involving hESCs were approved by the UTHealth Houston Stem Cell Research Oversight (SCRO) Committee (protocol #SCRO-23-01). For this protocol, all references to coated plates or wells refer to those prepared with hESC-qualified 3D basement membrane matrix.

1. Cell culture preparation

NOTE: Our lab uses H9 hESCs, but multiple hPSC lines have been used suc.......

Discussion

HIOs have been used as a model system for human intestinal development since the early 2010's and have become increasingly more complex since then. It is now possible to provide these constructs with an ENS, allowing for new opportunities in the study of normal development that may be applied to understanding and better treating a number of clinical gastrointestinal entities.

In vivo transplantation
ENCCs integrate into the mid-hindgut spher.......

Materials

Name	Company	Catalog Number	Comments
100x Non-Essential Amino Acids Solution (NEAA)	ThermoFischer Scientific	11140050
15 mL conical tubes	Thermo Scientific	12565269
Accutase	STEMCELL Technologies	07920	"enzymatic cell detachment reagent"
B-27 Supplement (50x), minus vitamin A	ThermoFischer Scientific	12587010	For ENCC
B-27 Supplement (50x), serum free	Gibco	17504044	For HIO
Bright-Line Hemacytometer	Hausser Scientific	3120
Corning Costar Ultra-Low Attachment Microplates	Fisher Scientific	07-200-601
Corning Flat-Bottom Plate 24-well, TC treated	VWR	29442-044
Corning Flat-Bottom Plate 3516 6 well	VWR	29442-042
Essential 6 Media	Thermo Fischer	A1516401
Essential 8 Media	Thermo Fischer	A2858501	Alternative stem cell media, used for ENCC plates.
Fine-tip forceps	Dumont	11223-20
Forma Steri-Cycle i160	Thermo Scientific	50145522
Gibco Advanced DMEM/F12	ThermoFischer Scientific	12634-010
Gibco HEPES 1 M	ThermoFischer Scientific	15630-080
Gibco Neurobasal Medium	ThermoFischer Scientific	21103-049
Glutagro, 200 mM, 100x	Corning	25-015-CI
Glutamax	ThermoFischer Scientific	35050061
H9 human ESC	Wicell International Stem Cell Bank	N/A
HyCloneTM FBS Defined	VWR	16777-002
LabGard Biological Safety Cabinet	Nuaire	Nu-430-400
Matrigel GFR Basement Membrane Matrix, Phenol Red-Free, LDEV-Free	Corning	356231	"Clear 3D basement membrane matrix"
Matrigel hESC-Qualified Matrix, LDEV-Free	Corning	354277	"3D basement membrane matrix"
Micropipettes	Eppendorf (100-1000, 20-200, 10-100, 2-20, 0.5-10, 0.1-2.5 uL)	2231300008
mTeSR 1	STEMCELL Technologies	85850	"stem cell media" Catalog number includes the 5x supplement, to be added in bulk in advance.
N-2 Supplement (100x)	Gibco	17502048	For HIO
N-2 Supplement, CTS (Cell Therapy Systems)	Thermo Fisher	A1370701	For ENCC. Slightly different formulation.
Nikon DS-Fi2 TS-100 microscope	Nikon	TS100
Noggin-conditioned media	Texas Medical Center Digestive Disease Center GEMS Core, Enteroid/Organoid Sub-core	N/A
Penicillin-Streptomycin (10,000 U/mL)	ThermoFischer Scientific	15140-122
Recombinant Human Activin A	Cell Guidance Systems	GFH6-100
Recombinant Human BMP-4	Fisher Scientific	314BP010
Recombinant Human EGF Protein, CF	ThermoFisher Scientific	236-EG-200
Recombinant Human FGF basic/FGF2 (146 aa) Protein	ThermoFischer Scientific	233-FB-010
Recombinant Human FGF-4	Peprotech	100-31
ReLeSR	STEMCELL Technologies	5872	"Stem cell dissociation reagent"
Retinoic acid	SIGMA	R2625-50MG
Rnase-free Microfuge tubes, 2 mL	Thermo Scientific	AM12425
RPMI 1640 Medium	ThermoFischer Scientific	11875093
R-Spondin conditioned media	Texas Medical Center Digestive Disease Center GEMS Core, Enteroid/Organoid Sub-core	N/A
SB 431542, Tocris Bioscience	Fisher Scientific	16-141-0
Sorvall ST 16R Centrifuge	Thermo Scientific
Standard Wide Orifice Pipettor Tips	VWR	89049-166
Stemolecule Chir99021 in Solution	Stemgent	04-0004-02
Sterile filter pipette tips	VWR (1000uL, 200uL, 10uL)	76322-154, 76322-150, 89174-520
Vitronectin XF	Stem Cell Technologies	7180	Alternative 3D basement membrane matrix, used for ENCC plates.