3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Summary

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Abstract

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:β-catenin gain-of-function (Smad4^KO:β-catenin^GOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 µm strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

Introduction

The intestinal crypts but not villi, form organoids when cultured in Matrigel or BME-R1 matrix. These organoids are self-organizing structures, often referred to as the "mini-gut" owing to the presence of the various differentiated lineages, progenitor, and stem cells present in the intestinal epithelium in vivo. The potential to form organoids from crypts is attributed to the presence of stem cells¹. The intestinal villi on the other hand consist only of differentiated cells, and hence cannot form organoids. However, mutations² or conditions that permit dedifferentiation of the villus epithelium may lea....

Protocol

All the mouse experiments conducted, including the use of Tamoxifen and euthanasia by cervical dislocation, had the approval of the Institutional Animal Care and Use Committee at Stevens Institute of echnology.

1. Mice

NOTE: The generation of Smad4^f/f; Catnb^lox(ex3)/⁺; Villin-Cre^ERT2 mice have been previously described³. Adult female mice between eight to twelve weeks of agewere used.

1. Induce the Smad4^KO:β-catenin^GOF mutation in the Smad4^f/f; Catnb^lox(ex3)/

Results

The determining factor for the success of the procedure is preventing crypt contamination. Organoid development from the villi (and not from any contaminating crypts) is ensured by confirming four major criteria: 1) ensuring the purity of the harvested villi by microscopic examination before and after plating the villi in BME-R1, 2) plating limited number of villi per well to allow visualization of all the plated villi individually, 3) onitoring the development of organoid daily; images of the time course show developmen.......

Discussion

This method can confirm the self-renewal capacity of dedifferentiating villi epithelium that acquire stem cell markers in vivo. The normal intestinal epithelium can give rise to organoids from the crypt but not the villi compartment, when cultured in 3D because of the presence of stem cells in the crypts¹. Thus, organoid formation from the dedifferentiated villi epithelium cultured in 3D cultures confirms stem cell formation from cell fate reversal. Reports on cell fate reversal in t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced DMEM F-12 media	Gibco	12634010
3,3-diaminobenzidine	Vector Labs	SK-4105
96 well U-bottom plate	Fisher Scientific	FB012932
ABC kit	Vector Labs	PK4001
Angled scissor	Fisher Scientific	11-999
Animal-Free Recombinant Human EGF	Peprotech	AF-100-15
B-27 Supplement (50X), minus vitamin A	Gibco	12587010
Bovine Serum Albumin (BSA) Protease-free Powder	Fisher Scientific	BP9703100
CD44 antibody	BioLegend	1030001
Cdx2 antibody	Cell Signaling	12306
Corn oil	Sigma-Aldrich	C8267-500ML
Corning 70-micron cell strainer	Life Sciences	431751
Cultrex Reduced Growth Factor Basement Membrane Extract, Type R1	R&D	3433-005-R1
Dissection scissors	Fisher Scientific	22-079-747
Forceps	Fisher Scientific	17-456-209
Glutamax (100X)	Gibco	35050-061
HEK 293-T cells expressing RSPO-1	Gift from Dr. Michael Verzi
HEPES (1M)	Gibco	15630-080
Histogel	Thermoscientific	HG-4000-012
Mesh filter	Fisher Scientific	07-201-431
Micrscope glass slide	VWR	89218-844
N-2 Supplement (100X)	Gibco	17502048
N-acetyl cysteine	Sigma-Aldrich	A9165
p200 Blunt tips	VWR	46620-642
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122
Primocin (50mg/mL)	Invivogen	ant-pm-1
Quality Biological Inc PBS (10X)	Fisher Scientific	50-146-770
Recombinant Murine Noggin	Peprotech	250-38
Signal diluent	Cell Signaling	8112L
Tamoxifen	Sigma-Aldrich	T5648-1G
6-well tissue culture plate	Fisher Scientific	50-146-770