The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

Summary

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5⁺) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

Abstract

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberkühn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate¹. Throughout the small intestine, Lgr5⁺ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5⁺ stem cells through several key ligands as Wnt3, EGF, and Dll1².

When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine³. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5⁺ and Paneth cells.

Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5⁺ cells to associate and form organoids when co-cultured. This approach, here referred to as "organoid reconstitution assay" (ORA), allows the genetic and biochemical modification of Paneth or Lgr5⁺ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

Introduction

The intestinal epithelium is the most rapidly self-renewing tissue in the mammalian body and, as such, has been the object of a plethora of studies aimed at the identification and functional characterization of the adult stem cells residing at the bottom of the crypt of Lieberkühn, earmarked by expression of the Lgr5 gene and dependent on canonical Wnt signals¹. Notably, Lgr5⁺ stem cells are flanked and supported by specialized niche cells, i.e. Paneth cells, which also depend on Wnt signaling for their maturation². Together, these two cell types underlie self-renewal of the int....

Protocol

All procedures were done according to local animal welfare laws and guidelines.

1. Preparation of Instruments, Culture Media, and Dishes

1. Autoclave 1 set of intestinal scissors, normal scissors, and forceps in a sterile container.
2. Place a 96-well (flat bottom) dish in an incubator at 37 °C.
3. Prepare 10 mL of complete culture medium with the reagents listed in table of materials.
4. Incubate the complete medium at 37 °C in a water bath.
5. Thaw reconstituted basement membrane by placing it in an ice bucket. The reconstituted basement membrane will become liquid at 4 °C .
6. Fi....

Results

The organoid reconstitution assay allows the separate functional analysis of the essential niche and stem cell components of the intestinal epithelium, here demonstrated by small interfering RNA (siRNA) of the Apc gene.

To achieve this aim, we first utilized fluorescent activated cell sorting (FACS) to separate 8000 Lgr5⁺ cells and 6000 Paneth cells from inbred C57BL6/J mice. In .......

Discussion

The ORA allows the refined functional analysis of the two essential components of the intestinal stem cell niche, namely Lgr5⁺ and Paneth cells. This approach has been previously employed by us and others with slight modifications^6,10,11. Here, we present the ORA procedure as a reproducible and standardized laboratory protocol. Also, we report on the effects of Apc downregulation in Lgr5

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced DMEM F12 *	Thermo Fisher Scientific	12634-010
Glutamax *	Thermo Fisher Scientific	35050061	Final concentration: 10 mM
Penicillin/streptomycin *	Thermo Fisher Scientific	15140122	Final concentration: 1%
Hepes *	Thermo Fisher Scientific	15630080	Final concentration: 10 mM
Recombinant murine EGF *	Thermo Fisher Scientific	PMG8041	Final concentration: 50 ng/ml
Recombinant murine Noggin *	Peprotech	250-38	Final concentration: 100 ng/ml
y27632 *	Sigma	Y0503	Final concentration: 10 µM
Jagged-1 *	Anaspec Bio	AS-61298	Final concentration: 1 µM
Recombinant murine R-spondin *	R&D systems	3474-RS-050	Final concentration: 1 mg/ml
Flexitube Gene solution siRNA APC	Qiagen	GS11789	Final concentration: 100 nM
Lipofectamine 2000	Thermo Fisher Scientific	11668019
CD24 APC	Biolegend	101814
c-kit PE	Biolegend	105808
BV 421 CD31	Biolegend	102424
BV 421 CD45	Biolegend	103134
BV421 TER119	Biolegend	116234
HBSS	Thermo Fisher Scientific	14180046
B6.129P2-Lgr5tm1(cre/ERT2)Cle/J	Jackson Laboratories	008875
Low binding tubes	Eppendorf	Z666548-250EA
Matrigel	Corning	356230
* The combination of these reagents constitutes the complete culture medium