The authors would like to thank Mark Bishop, Mathew Zverev, Victoria Marsh-Durban, Adam Blackwood and Sylvie Robine. This work was funded by a programme grant from Cancer Research UK.

<ol>
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The authors have nothing to disclose.

Using conditional cre-lox transgenic mice to dissect the function of genes and cells is a commonly used approach. These models have been used with great success in the intestine to identify and characterize the stem cells2,4-6 and understand their role in disease25. To fully exploit these models requires a comprehensive characterization of the system to enable data to be interpreted correctly. A complete understanding of these systems is difficult to achieve due to genes rarely being specific to a solitary cell type or location, a lack of biological knowledge and inefficiency of the systems used to induce Cre expression. The methods described here demonstrate how we overcome these issues through experimental design and application of existing knowledge. Although we used these methods to answer a specific research question the techniques presented here are generic and can be exploited for any research investigating the murine intestine.
Preparation of Intestinal Tissue
The crucial step for ensuring robust results is the harvesting and processing of the tissue, which needs to be processed in a timely manner and fixation protocols strictly adhered to. As almost all significant issues downstream can be attributed to artefacts associated with the tissue drying out and/or incomplete fixation. Timing is crucial to prevent degradation of tissue architecture and/or nucleic acids and proteins. Incomplete or overzealous fixation can result in loss of histochemical resolution. Incomplete fixation due to insufficient time or sections too thick to allow fixative penetration can result in loss of resolution within the intestinal crypts that can be observed as a &#8220;tide mark&#8221; upon IHC analysis. Further it is crucial that fixation does not extend for too long, as nuclear &#946;-catenin can diffuse out of the nucleus unless immediately processed and wax embedded following formalin fixation.
Role of Paneth Cells in the ISC Niche
The data presented here effectively show the importance of Paneth cells in crypt regeneration in the adult intestine following ISC loss. However there remained the possibility that Ah-cre spares a population of ISCs that vil-Cre-ERT2 targets. Tian et al.26 elegantly demonstrated that the Lgr5hi ISCs are replaced by a population of Lgr5lo reserve ISCs. It now seems likely that these ISCs are spared in the Ah-cre system due to the reserve population having been identified as secretory cell precursors6,7. The importance of the mature Paneth cell in supporting these secretory cell precursors when required to revert to an ISC state remains to be answered. As Paneth cells constitute the ISC niche8 and play roles in regulating the ISC responses to calorie intake27 and inflammation28 it remains likely that their nursing functions will extend to their own precursors.
New Approaches and Technologies to Effectively Model Human Colorectal Cancer
The discovery of the ISC led to the identification of genes which are now being used to generate new mouse models for investigating the roles of genes and cells in intestinal biology and disease, reviewed by Clarke et al9. The only limitations to this technique is the identification of genes to express the Cre protein. Currently ISCs are routinely investigated using conditional transgenic mice based on the Lgr5 gene expression pattern. Mice which express Cre from the Lgr5 promoter have been used to delete Apc, the gene most commonly mutated in colorectal cancer (CRC), demonstrating the ISC as the cell of origin25. Selectively deleting other CRC genes in these cells is providing insight into disease progression and spread e.g. PTEN29. Further insight into ISC function is being retrieved by specifically ablating Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus26. Other strategies use the Tet-O system which enables ongoing reversible expression of mutant proteins30. Using these tools to modify gene(s) in different cells31 and locations32,33 is used to understand how cancer initiate, progress and metastasize34. Alternatively mutagenesis using the sleeping beauty transposon system is identifying new drivers of CRC. The continuing development of mice, techniques and genetic alteration strategies is continuing to develop more patient relevant models.
New methods have been developed for characterization of the intestinal epithelia and ISCs. Characterization of the ratio of epithelial cell types can be achieved using flow cytometry based on the differential expression of lectin and CD2435. Potentially the biggest progress in understanding ISC biology and their role in disease will be made using the ex vivo organoid culture system36. This system allows normal and malignant ISCs to culture in 3D, where they replicate and differentiate in a more physiologically relevant way. It is hoped that these will enable direct testing of drugs on patient samples in vitro, paving the way for personalized medicine37.

The luminal surface of the mammalian intestine features repeating units of crypts and finger like projections, termed villi, which protrude into the lumen. This surface is a continuous sheet of epithelia which undergoes complete self-renewal approximately every 3 - 4 days1. This dynamic tissue is supported by a population of rapidly cycling stem cells (ISCs; also known as crypt base columnar cells), which were initially identified by their expression of the Lgr5 gene2,3. These cells exist in a specialized niche at the bottom of the crypts of Lieberkuhn. Initially, the discovery that ISCs were rapidly cycling was discordant with the prevailing idea that a stem cell was quiescent in nature. Previous to the identification of the Lgr5+ ISC it was postulated that a population of quiescent label retaining cells at the +4 position, relative to the base of the crypt, were the ISCs1. Recent research has now reconciled these observations by demonstrating that primarily there is a pool of equipotent cycling ISCs in each crypt whose fate are regulated by its neighbors4,5. In the event they are lost these can be replaced by quiescent cells that ordinarily are committed to the secretory lineage but can revert to ISCs if the ISC population is damaged6.
ISC neighbors can either be ISCs or their daughter cells. The ISCs produce na&#239;ve daughter cells which multiply and differentiate into the specialized cell types that comprise the epithelial sheet which lines the intestinal lumen1. The goblet, enteroendocrine, enterocytes, tuft and M cells migrate upwards to the luminal surface where they provide various absorptive and regulatory functions, however, the Paneth cells remain at the bottom of the crypt where they exist intermingled with the ISCs. In recent years it has been demonstrated that a proportion of the na&#239;ve daughter cells destined for a secretory lineage are quiescent label retaining Lgr5lo cells capable of reverting to an ISC upon injury6,7.
Due to its importance in crypt regeneration a priority was placed on understanding the interactions between the ISCs and its neighbors, particularly the Paneth cells. The Paneth cells play a crucial role in the niche which supports the ISCs8. In addition to bactericidal products the Paneth cells produce signaling molecules that activate the pathways which govern ISC renewal or differentiation. Previous studies showed that the Lgr5+ ISCs could only exist when they could compete for essential niche signals provided by their daughter Paneth cells8. These studies examined the role of Paneth cells on normal Lgr5+ ISCs and not in a situation where they are damaged and require replenishment from an Lgr5lo population.
To understand intestinal biology and model disease we examine the functional role of cells and/or genes using transgenic mouse models9,10. Frequently these models utilize cre-lox technology to conditionally modify gene(s)9,10. Cre (Causes recombination) recombinase is a site specific recombinase of the integrase family, isolated from bacteriophage P1. Cre catalyses site specific recombination between defined 34 bp &#8216;Lox P&#8217; (locus &#967; of crossover P1) sites. Mice are genetically engineered to contain LoxP sites that flank regions of interest which upon expression of Cre recombinase are excised. Linking the expression of the Cre gene to a cell or developmental specific promoter allows for alteration to be made in a spatial fashion9,10, this is especially useful in overcoming embryonic lethal mutations. Further linking the Cre expression to a receptor pathway, that can be activated artificially, permits temporal alterations.
Using this technology we inactivated the CatnB gene11 in the intestinal epithelia. &#946;-catenin, the CatnB gene product, is a key regulator of the canonical Wnt signaling pathway which governs ISC homeostasis. Two previous studies using this strategy produced conflicting results12,13. The study by Fevr et al.12, demonstrated loss of stem cells and intestinal homeostasis. Whereas the Ireland et al.14study reported that following a reduction in cell viability the crypt-villus axis was repopulated from wild type cells expressing CatnB. The major difference in these studies was the promoter used to express Cre in the intestinal epithelia. The Fevr et al., study used the villin gene promoter linked to the estrogen receptor which can be activated by administering tamoxifen (vil-Cre-ERT2)15,16. In contrast Ireland et al., utilized the promoter element of the rat cytochrome P450A1 (CYP1A1) gene to drive Cre expression in response to the xenobiotic &#946;-naphthoflavone (Ah-cre). The characteristics of these different systems generated two hypotheses to account for these different observations. The first that CatnB is more efficiently deleted in the ISC using the vil-Cre-ERT2 system compared to the Ah-cre, thereby reducing the number of ISCs to sub-repopulation levels. Alternatively it was due to differential CatnB deletion in the differentiated cell population. The vil-Cre-ERT2 system targets all epithelial cells of the crypt and villus whereas the Ah-cre system only targets the non-Paneth cells of the ISC niche and crypt. These systems provided ideal tools for examining the behavior of the ISCs and their interaction with the Paneth cells. Here we present several detailed protocols based on how we used these systems to determine that Paneth cells play a crucial role in mediating the intestinal response to injury17.

<table><tbody><tr><td>Acetate buffer</td><td>*</td><td>*</td><td>To make 100 ml: 4.8 ml 0.2 M Acetic acid, 45.2 ml 0.2 M Sodium acetate &amp;amp; 50 ml distilled water</td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>C/0400/PB17</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma</td><td>A6404</td><td></td></tr><tr><td>Acetic anhydride solution</td><td>*</td><td>*</td><td>2 M Acetic anhydride in 0.1 M triethanolamine hydrochloride</td></tr><tr><td>Alcian Blue</td><td>Sigma</td><td>A5268</td><td></td></tr><tr><td>Alcian Blue pH 2.5</td><td>*</td><td>*</td><td>To make 500 ml: 15 ml acetic acid, 5 g Alcian Blue &amp;amp; 485 ml distilled water</td></tr><tr><td>anti-digoxigenin alkaline phosphatase conjugated antibody</td><td>Abcam</td><td>ab119345</td><td></td></tr><tr><td>B(beta)-Naphthoflavone</td><td>Sigma</td><td>N3633</td><td>BNF, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &amp;ndash; store at -20 &amp;deg;C between uses, do not reheat more than twice.</td></tr><tr><td>Bloxall</td><td>Vector Labs</td><td>SP-6000</td><td></td></tr><tr><td>BM purple</td><td>Roche</td><td>11442074001</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A4503</td><td>Bovine serum albumin</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C/4920/17</td><td></td></tr><tr><td>Citrate Buffer/Antigen Unmasking Solution</td><td>Vector Labs</td><td>H-3300</td><td></td></tr><tr><td>Corn oil</td><td>Sigma</td><td>C8627</td><td></td></tr><tr><td>Demucifiying solution</td><td>*</td><td>*</td><td>For 500 ml: 50 ml glycerol, 50 ml Tris 0.1M pH8.8, 100 ml EtOH, 300 ml saline (0.9% NaCl in water), DTT 1.7 g.&amp;nbsp; Demucifying solution can be made in advance and stored, but DTT sholud be added just before incubation (340 mg/100 ml).</td></tr><tr><td>DEPC treated water</td><td>Life Technologies</td><td>750023</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>101509944</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>O3690</td><td>0.5M</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>E/0650DF/17</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td>3000917</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775</td><td></td></tr><tr><td>Formalin&amp;nbsp;</td><td>Sigma</td><td>SLBL11382V</td><td>Neutral buffered formalin</td></tr><tr><td>Formamide</td><td>Sigma</td><td>F5786</td><td></td></tr><tr><td>Glutaraldehye</td><td>Sigma</td><td>G6257</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>216763</td><td></td></tr><tr><td>Haematoxylin</td><td>Raymond A Lamb</td><td>12698616</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-053</td><td>HBSS (-MgCl2+; -CaCl2)</td></tr><tr><td>Hybridisation buffer</td><td>*</td><td>*</td><td>&amp;nbsp;5&amp;times; SSC, 50% formamide, 5% SDS, 1 mg/ml heparin, 1 mg/ml calf liver tRNA</td></tr><tr><td>Hydroquinone</td><td>Sigma</td><td>H9003</td><td></td></tr><tr><td>ImmPACT DAB Peroxidase</td><td>Vector Labs</td><td>SK-4105</td><td></td></tr><tr><td>Immpress HRP Anti-Mouse IgG Kit</td><td>Vector Labs</td><td>MP-7402</td><td></td></tr><tr><td>Immpress HRP Anti-Rabbit IgG Kit</td><td>Vector Labs</td><td>MP-7401</td><td></td></tr><tr><td>Intestinal tissue powder</td><td>*</td><td>*</td><td>The small intestines of 5 adult mice were combined and homogenised in the minimum volume of ice cold PBS. 4 volumes of ice cold acetone were added to the homogenised intestine, which was mixed thoroughly and incubated on ice for 30 min. This was centrifuged and the pellet was washed using ice cold acetone. This was further centrifuged and the resulting pellet spread onto filter paper and allowed to dry. Once thoroughly dry the material was ground to a fine powder using a pestle and mortar.&amp;nbsp;</td></tr><tr><td>K-ferricyanide</td><td>Sigma</td><td>P-3667</td><td></td></tr><tr><td>K-ferrocyanide</td><td>Sigma</td><td>P3289</td><td></td></tr><tr><td>Levamisole</td><td>Sigma</td><td>L0380000</td><td></td></tr><tr><td>Methacarn</td><td>*</td><td>*</td><td>60% Methanol:30% Chloroform:10% Acetic acid</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>M/4000/17</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Normal goat serum</td><td>Vector Labs</td><td>S-1012</td><td>NGS</td></tr><tr><td>Normal rabbit serum</td><td>Dako</td><td>X0902</td><td>NRS</td></tr><tr><td>NTMT</td><td>*</td><td>*</td><td>100 mM NaCl, 100 mM Tris HCl, 50 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.1% Tween20, 2 mM Levamisole</td></tr><tr><td>PAP pen</td><td>Vector</td><td>H-400</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>PBT</td><td>*</td><td>*</td><td>0.5 M NaCl, 10 mM TrisHCL pH7.5, 0.1% Tween 20</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td>100x solutiuon.</td></tr><tr><td>Phosphate buffered saline (10x)</td><td>Fisher Scientific</td><td>BP3994</td><td>Dilluted 1:10 with distilled water to make 1x</td></tr><tr><td>PLL slides</td><td>Sigma</td><td>P0425-72EA</td><td>Poly-L-lysine microscope slides</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P2308</td><td></td></tr><tr><td>Proteinase k solution</td><td>*</td><td>*</td><td>Dilute Proteinase K at 200 &amp;micro;g/ml in 50 mM Tris, 5 mM EDTA.</td></tr><tr><td>Ralwax</td><td>BDH</td><td>36154 7N</td><td></td></tr><tr><td>Reducer Solution</td><td>*</td><td>*</td><td>To make 100 ml: 1 g Hydroquinone, 5 g sodium sulphite &amp;amp; 100 ml distilled water</td></tr><tr><td>RnaseA</td><td>Sigma</td><td>R6148</td><td></td></tr><tr><td>Saline</td><td>*</td><td>*</td><td>0.9% NaCl in distilled water</td></tr><tr><td>SDS</td><td>Sigma</td><td>I3771</td><td></td></tr><tr><td>Sheep serum</td><td>Sigma</td><td>S3772</td><td></td></tr><tr><td>Silver nitrate</td><td>Sigma</td><td>S/1240/46</td><td></td></tr><tr><td>Silver solution</td><td>*</td><td>*</td><td>To make 100 ml: 10 ml Acetate buffer, 87 ml distilled water, 3 ml 1% silver nitrate</td></tr><tr><td>Sodium acetate</td><td>Fisher Scientific</td><td>S/2120/53</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S6753</td><td>NaCl</td></tr><tr><td>Sodium sulfite</td><td>Sigma</td><td>239321</td><td></td></tr><tr><td>SSC</td><td>Sigma</td><td>93017</td><td>20x saline sodium citrate</td></tr><tr><td>Surgical tape</td><td>Fisher Scientific</td><td>12960495</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td><td>TAM, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &amp;ndash; store at -20 &amp;deg;C between uses, do not reheat more than twice.</td></tr><tr><td>TBS/T</td><td>Cell Signalling</td><td>#9997</td><td></td></tr><tr><td>Triethanolamine hydrochloride</td><td>Sigma</td><td>T1502</td><td></td></tr><tr><td>Tris-HCL</td><td>Invitrogen</td><td>15567-027</td><td></td></tr><tr><td>Tween20</td><td>Sigma</td><td>TP9416</td><td></td></tr><tr><td>VectaMount</td><td>Vector Labs</td><td>H-5000</td><td></td></tr><tr><td>VectaShield Hardset mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1500</td><td></td></tr><tr><td>Vectastain ABC Kit</td><td>Vector Labs</td><td>PK-4001</td><td></td></tr><tr><td>X-gal</td><td>Promega</td><td>V3941</td><td></td></tr><tr><td>X-gal fixative</td><td>*</td><td>*</td><td>2% formaldehyde, 0.1% glutaraldehyde in 1x PBS</td></tr><tr><td>X-gal stain</td><td>*</td><td>*</td><td>X-gal stain; 200 &amp;#956;l X-gal (A) in 50 ml solution B (0.214 g MgCl2, 0.48 g K-ferricyanide, 0.734 g K-ferrocyanide in 500 ml PBS).&amp;nbsp; Solution B can be made up oin advance and stored at 4 &amp;deg;C</td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X/0200/21</td><td></td></tr></tbody></table>

protocols for analyzing the role of paneth cells in regenerating the murine intestine using conditional cre-lox mouse models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Watch this Scientific Journal Video about Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models at JoVE.com

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

protocols-for-analyzing-role-paneth-cells-regenerating-murine

Research

JoVE Journal

Developmental Biology

17.6K Views.  Cardiff University.  Intestinale epitheliale stamcellen (ISC) worden vermengd met Paneth cellen. Deze cellen zijn gedifferentieerd nageslacht van de ISC, die ISC ondersteunen en antibacteriële bescherming. Hier laten we zien hoe we gebruikten conditionele transgene muismodellen aangetoond dat Paneth cellen spelen een cruciale rol bij het handhaven van de intestinale epitheel. 

Video: Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

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

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 Lieberk&#252;hn, 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 &#34;organoid reconstitution assay&#34; (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.

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

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.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Bioengineering

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Immunology and Infection

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Biology

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

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