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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 lead to stem cells in the villi^2,3. This fate change resulting in stemness in the villi epithelium can be confirmed by plating the dedifferentiating villus epithelium in 3D matrix to determine their organoid-forming potential as an indicator of de-novo stemness in the villus epithelium. Hence, the critical aspect of this procedure is to ensure absence of crypt contamination.

The Smad4^KO:β-catenin^GOF conditional mutation causes dedifferentiation in the intestinal epithelium marked by the expression of proliferation and stem cell markers in the villi, and eventually the formation of crypt-like structures in the villi referred to as ectopic crypts The presence of stem cells these dedifferentiated villi was determined by the expression of stem cell markers in the ectopic crypts (in vivo) and the ability of the mutant villi to form organoids when plated Matrigel³. The below mentioned procedure elaborates the methodology used to confirm the stemness of the dedifferentiating intestinal epithelium in the Smad4^KO:β-catenin^GOF mutant mice. A key feature of this methodology for isolating villi was the use of scraping of the intestinal lumen, as opposed to the EDTA chelation method⁴. Unlike in the EDTA chelation method, villi isolation by scraping retains majority of the underlying mesenchyme and allows adjust the pressure of scraping to yield villi without tethered crypts. The pressure of scraping is subjective to the operator, hence, the optimal pressure to yield villi without crypts tethered must be determined empirically by the operator. The critical aspect of this procedure is to ensure the absence of crypt contamination by microscopic examination of the villi both before and after plating in the BME-R1 matrix.

Intestinal villi are scraped off the intestinal lumen with glass slides and placed in a 70 µm filter and washed with PBS to get rid of loose cells or crypts, if any, prior to plating in BME-R1 matrix. The method stresses on the following criteria to avoid crypt contamination: a) confining the villi harvest the proximal half of the duodenum where the villi are the longest, b) minimizing the number of villi-yielding scrapes, c) washing the filter containing the villi through a series of PBS in a six-well dish, and d) confirming the absence of crypt contamination by microscopic examination prior to and after plating in BME-R1 matrix. Villi isolation by scraping, rather than by EDTA chelation, prevents the complete loss of the underlying mesenchyme that may provide the niche signals^{5, 6, 7, 8}, if required, for organoid initiation from the villus epithelium.

Protokół

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)/⁺; Villin-Cre^ERT2 with intraperitoneal injection of Tamoxifen in corn oil for four consecutive days.
2. Sacrifice the miceby cervical dislocation ten days after the first tamoxifen injection.
3. Spray the abdomen with 70% ethanol to prevent mouse fur getting into the peritoneal cavity.
4. Open the abdominal cavity with dissection scissors to expose the intestine. Isolate the intestine with the aid of the scissors and forceps.
   ​NOTE: Concomitant loss of Smad4 along with gain of function mutation of β-catenin (Smad4^KO;β-catenin^GOF) in intestinal epithelium was attained by injecting^Smad4f/f; Catnb^lox(ex3)/⁺; Villin-Cre^ERT2 mice every day for four consecutive days with 0.05 g Tamoxifen/kg body weight in corn oil. These mice are harvested ten days after the first tamoxifen injection to ensure the presence of cells expressing stem cell-associated markers in the dedifferentiating villi.

2. Duodenum isolation and preparation

1. Dissect out the proximal half of the duodenum.
2. Flush the duodenum with 5 mL of ice-cold PBS in a 10 mL syringe to clear the luminal content.
3. Open the duodenum longitudinally with an angled scissor and lay the duodenum flat on a 15 cm Petri plate on ice with the lumen of the duodenum facing the operator.

3. Villi isolation by scraping

1. Prior to beginning the scraping, place a 70 µm mesh strainer in one of the wells of a 6-well tissue culture plate. Fill all the wells with 4 mL of 1x PBS and place the 6-well tissue culture plate on ice (Figure 1).
2. Scrape the villi as follows using two microscopic glass slides: one to hold the duodenum down and the other to scrape (Figure 1B1).
   1. Scrape the luminal side of the duodenum superficially to-and-fro twice to remove the mucus. Apply pressure such that this step removes the mucus layer, but not the villi.
   2. Scrape the duodenum again, to-and-fro twice applying the same pressure as in 3..1, during which villi can be seen collecting on the slides (Figure 1B2). This is the optimal pressure (which must be determined empirically by the operator) to yield the villi without the crypts tethered.
3. Use a 1 mL transfer pipet containing PBS to transfer the villi (that are collected on the slide from step 3.2.2.) to a 70 µm mesh strainer placed in a 6-well dish. The villi are collected thus, after every scrape (Figure 1B2).
4. Wash the villi collected in the 70 µm strainer (from step 3.3) by transferring the strainer (with the villi) through a series of wells in a 6-well dish containing cold PBS (~ 4 mL/well). This is to remove loose crypts, if any.
5. Using a p1000 pipet, transfer the villi suspension in PBS (~ 3 mL) from the 70 µm strainer to a new 15 mL tube on ice.
6. Use a 0.1% BSA coated blunt-ended p200 pipet tip to aspirate 50 µL volume of the villi suspension onto a glass slide. Count the number of villi in the 50 µL droplet under 4x magnification to determine the concentration of villi in the PBS suspension. For example, if there are 10 villi in the 50 µL suspension examined, then the villi concentration is 0.2 villi/µL. This is also the time to confirm the absence of tethered crypts.
7. Calculate the volume of villi suspension required to plate at a concentration of 0.5 villi/µL of BME-R1 matrix. Add an extra 100 µL to account for pipetting error and the volume required for microscopic examination required to ensure the purity of the villi.
   ​CAUTION: The pressure used in the first two scrapes, that removes the mucus but not the villi, should be used in the subsequent scrapes during which villi will be released. Limit the number of to-and-fro scrapings to two after the villi release is first observed. This measure avoids release of villi with the crypts tethered. It is essential to microscopically evaluate the villi to ensure the absence of the tethered crypts.

4. Plating of villi on BME-R1 matrix

1. Using a 0.1% BSA coated p200 blunt-ended pipet tip, transfer to a microcentrifuge tube the volume of villi (from step 3.6) required to plate at a density of ~ 6 villi per well in 12.5 µL of BME-R1 matrix. The use of BSA-coated blunt-ended tip at this point ensures that the villi, which are too large for a pointed tip are aspirated without being blocked or lost from being stuck to the sides of the tip.
2. Spin down the villi for 2 min at 200 x g in a refrigerated centrifuge (4 °C) and remove the supernatant.
3. Repeat step 4.2 to remove any residual PBS and proceed to the next step under a laminar flow-hood.
4. Resuspend the villi pellet gently in the required amount of cold BME-R1 thawed on ice.
5. Using a p20 pipet, plate 12.5 µL/well of the villi in BME-R1 matrix in 3D in a prewarmed 96-well U-bottom plate.
6. Incubate the plate in a tissue culture incubator at 37 °C for 15 minutes to allow solidification of the BME-R1 matrix.
7. Add 125 µL/well of pre-warmed ENR (Epidermal growth factor/Noggin/R-spondin1) media: advanced DMEM F-12 media, supplemented with 1x Penicillin: Streptomycin, 10 mM HEPES, Glutamax, 50 ng/mL EGF, 100 ng/mL Noggin, 5% conditioned media from HEK293-T cells expressing R-Spondin1, 1x N2, 1x B27, 1 mM N-acetyl cysteine, and 0.05 mg/mL Primocin.
8. Incubate the plated villi in a tissue culture incubator maintained at 37 °C with 5% CO₂, and change the media every other day.
9. Discard any well in which organoids appear before two days of plating, since the earliest time point at which villi-derived organoids are expected is after two days.
   NOTE: Any villi that has half or more than half the length of villi are counted as one villus. Unlike crypt-derived organoids, villi-derived organoids are not expected within 24 hours after plating. The organoid-initiating villi appear to be darkening and shrinking prior to formation of organoids. Hence, any wells with organoids developing within a day of plating should be discarded to avoid the plausibility of having derived from plausible crypt contamination. The methodology used to produce the conditioned media is available upon request.

Wyniki

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...

Dyskusje

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...

Materiały

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