Quantitative classification of chromatin dynamics reveals regulators of intestinal stem cell differentiation

Raab JR, Tulasi DY, Wager KE, Morowitz JM, Magness ST, Gracz AD

Development. 2020 Jan 3;147(1). PMID: 31862843

Abstract

Intestinal stem cell (ISC) plasticity is thought to be regulated by broadly permissive chromatin shared between ISCs and their progeny. Here, we have used a Sox9EGFP reporter to examine chromatin across ISC differentiation. We find that open chromatin regions (OCRs) can be defined as broadly permissive or dynamic in a locus-specific manner, with dynamic OCRs found primarily in loci consistent with distal enhancers. By integrating gene expression with chromatin accessibility at transcription factor (TF) motifs in the context of Sox9EGFP populations, we classify broadly permissive and dynamic chromatin relative to TF usage. These analyses identify known and potential regulators of ISC differentiation via association with dynamic changes in chromatin. Consistent with computational predictions, Id3-null mice exhibit increased numbers of cells expressing the ISC-specific biomarker OLFM4. Finally, we examine the relationship between gene expression and 5-hydroxymethylcytosine (5hmC) in Sox9EGFP populations, which reveals 5hmC enrichment in absorptive lineage-specific genes. Our data demonstrate that intestinal chromatin dynamics can be quantitatively defined in a locus-specific manner, identify novel potential regulators of ISC differentiation and provide a chromatin roadmap for further dissecting cis regulation of cell fate in the intestine.

Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach

Min J, Vega PN, Engevik AC, Williams JA, Yang Q, Patterson LM, Simmons AJ, Bliton RJ, Betts JW, Lau KS, Magness ST, Goldenring JR, Choi E

Nat Commun. 2019 Dec 5;10(1):5549. PMID:31804471

Abstract

Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However, the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines, derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib, a MEK inhibitor, which is a downstream mediator of Kras signaling. Here, we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However, the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells, which exhibited different clonogenic potentials. Therefore, Kras activation controls cellular dynamics and progression to dysplasia, and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages.

Interferon-λ3 Promotes Epithelial Defense and Barrier Function Against Cryptosporidium parvum Infection

Ferguson SH, Foster DM, Sherry B, Magness ST, Nielsen DM, Gookin JL

Cell Mol Gastroenterol Hepatol. 2019;8(1):1-20.  Epub 2019 Mar 5. PMID: 30849550

Abstract

BACKGROUND & AIMS: The epithelial response is critical for intestinal defense against Cryptosporidium, but is poorly understood. To uncover the host strategy for defense against Cryptosporidium, we examined the transcriptional response of intestinal epithelial cells (IECs) to C parvum in experimentally infected piglets by microarray. Up-regulated genes were dominated by targets of interferon (IFN) and IFN-λ3 was up-regulated significantly in infected piglet mucosa. Although IFN-λ has been described as a mediator of epithelial defense against viral pathogens, there is limited knowledge of any role against nonviral pathogens. Accordingly, the aim of the study was to determine the significance of IFN-λ3 to epithelial defense and barrier function during C parvum infection. METHODS: ​The significance of C parvum-induced IFN-λ3 expression was determined using an immunoneutralization approach in neonatal C57BL/6 mice. The ability of the intestinal epithelium to up-regulate IFN-λ2/3 expression in response to C parvum infection and the influence of IFN-λ2/3 on epithelial defense against C parvum invasion, intracellular development, and loss of barrier function was examined using polarized monolayers of a nontransformed porcine-derived small intestinal epithelial cell line (IPEC-J2). Specifically, changes in barrier function were quantified by measurement of transepithelial electrical resistance and transepithelial flux studies. RESULTS: Immunoneutralization of IFN-λ2/3 in C parvum-infected neonatal mice resulted in a significantly increased parasite burden, fecal shedding, and villus blunting with crypt hyperplasia during peak infection. In vitro, C parvum was sufficient to induce autonomous IFN-λ3 and interferon-stimulated gene 15 expression by IECs. Priming of IECs with recombinant human IFN-λ3 promoted cellular defense against C parvum infection and abrogated C parvum-induced loss of barrier function by decreasing paracellular permeability to sodium. CONCLUSIONS: These studies identify IFN-λ3 as a key epithelial defense mechanism against C parvum infection.

Preservation of reserve intestinal epithelial stem cells following severe ischemic injury

Gonzalez LM, Stewart AS, Freund J, Kucera CR, Dekaney CM, Magness ST, Blikslager AT

Am J Physiol Gastrointest Liver Physiol. 2019 Apr 1;316(4):G482-G494.  Epub 2019 Feb 4. PMID: 30714814

Abstract

Intestinal ischemia is an abdominal emergency with a mortality rate >50%, leading to epithelial barrier loss and subsequent sepsis. Epithelial renewal and repair after injury depend on intestinal epithelial stem cells (ISC) that reside within the crypts of Lieberkühn. Two ISC populations critical to epithelial repair have been described: 1) active ISC (aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 positive, sex determining region Y-box 9 positive) and 2) reserve ISC [rISC; less proliferative; homeodomain only protein X (Hopx)+]. Yorkshire crossbred pigs (8-10 wk old) were subjected to 1-4 h of ischemia and 1 h of reperfusion or recovery by reversible mesenteric vascular occlusion. This study was designed to evaluate whether ISC-expressing biomarkers of aISCs or rISCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative responses. Our data demonstrate that, following 3-4 h ischemic injury, aISC undergo apoptosis, whereas rISC are preserved. Furthermore, these rISC are retained ex vivo in spheroids in which cell populations are enriched in the rISC biomarker Hopx. These cells appear to go on to provide a proliferative pool of cells during the recovery period. Taken together, these data indicate that Hopx+ cells are resistant to injury and are the likely source of epithelial renewal following prolonged ischemic injury. It is therefore possible that targeting reserve stem cells will lead to new therapies for patients with severe intestinal injury. NEW & NOTEWORTHY The population of reserve less-proliferative intestinal epithelial stem cells appears resistant to injury despite severe epithelial cell loss, including that of the active stem cell population, which results from prolonged mesenteric ischemia. These cells can change to an activated state and are likely indispensable to regenerative processes. Reserve stem cell targeted therapies may improve treatment and outcome of patients with ischemic disease.

IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model

Zwarycz B, Gracz AD, Rivera KR, Williamson IA, Samsa LA, Starmer J, Daniele MA, Salter-Cid L, Zhao Q, Magness ST

Cell Mol Gastroenterol Hepatol. 2018 Jul 4;7(1):1-17. eCollection 2019. PMID: 30364840

Abstract

Crohn's disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn's disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal. METHODS: A screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo. RESULTS: High IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal-associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs. CONCLUSIONS: 

Increased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.

A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies

Gunasekara DB, Speer J, Wang Y, Nguyen DL, Reed MI, Smiddy NM, Parker JS, Fallon JK, Smith PC, Sims CE, Magness ST, Allbritton NL

Anal Chem. 2018 Nov 20;90(22):13331-13340. Epub 2018 Oct 30. PMID:  30350627

Abstract

Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability ( Papp) of <5 × 10-7 cm s-1, while propranolol demonstrated a Papp of 9.69 × 10-6 cm s-1. Rh123 was transported in a luminal direction ( Papp,efflux/ Papp,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo.

Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity

Jin Y, Ibrahim D, Magness ST, Blikslager AT

Am J Physiol Gastrointest Liver Physiol. 2018 Dec 1;315(6):G966-G979. Epub 2018 Oct 4. PMID: 30285466

Abstract

Adherens junctions (AJs), together with tight junctions (TJs), form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ, membrane-bound E-cadherin binds β-catenin, which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex, chloride channel protein-2 (ClC-2), that we have used to study TJ regulation. In this study, we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation, whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore, the absence of ClC-2 resulted in redistribution of AJ proteins and increased β-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased, associated with β-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of β-catenin-associated signaling. NEW & NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures, including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating β-catenin signaling.

Quantitative Analysis of Intestinal Stem Cell Dynamics Using Microfabricated Cell Culture Arrays

Samsa LA, Williamson IA, Magness ST

Methods Mol Biol. 2018;1842:139-166. PMID: 30196407

Abstract

Regeneration of intestinal epithelium is fueled by a heterogeneous population of rapidly proliferating stem cells (ISCs) found in the base of the small intestine and colonic crypts. ISCs populations can be enriched by fluorescence-activated cell sorting (FACS) based on expression of combinatorial cell surface markers, and fluorescent transgenes. Conventional ISC culture is performed by embedding single ISCs or whole crypt units in a matrix and culturing in conditions that stimulate or repress key pathways to recapitulate ISC niche signaling. Cultured ISCs form organoid, which are spherical, epithelial monolayers that are self-renewing, self-patterning, and demonstrate the full complement of intestinal epithelial cell lineages. However, this conventional "bulk" approach to studying ISC biology is often semiquantitative, low throughput, and masks clonal effects and ISC phenotypic heterogeneity. Our group has recently reported the construction, long-term biocompatibility, and use of microfabricated cell raft arrays (CRA) for high-throughput analysis of single ISCs and organoids. CRAs are composed of thousands of indexed and independently retrievable microwells, which in combination with time-lapse microscopy and/or gene-expression analyses are a powerful tool for studying clonal ISC dynamics and micro-niches. In this protocol, we describe how CRAs are used as an adaptable experimental platform to study the effect of exogenous factors on clonal stem cell behavior.

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology

Williamson IA, Arnold JW, Samsa LA, Gaynor L, DiSalvo M, Cocchiaro JL, Carroll I, Azcarate-Peril MA, Rawls JF, Allbritton NL, Magness ST

Cell Mol Gastroenterol Hepatol. 2018 May 22;6(3):301-319. eCollection 2018. PMID: 30123820

Abstract

BACKGROUND & AIMS: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. METHODS: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. RESULTS: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. CONCLUSIONS: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.

Reserve Stem Cells in Intestinal Homeostasis and Injury

Bankaitis ED, Ha A, Kuo CJ, Magness ST

Gastroenterology. 2018 Nov;155(5):1348-1361.  Epub 2018 Aug 15. Review. PMID: 30118745

Abstract

Renewal of the intestinal epithelium occurs approximately every week and requires a careful balance between cell proliferation and differentiation to maintain proper lineage ratios and support absorptive, secretory, and barrier functions. We review models used to study the mechanisms by which intestinal stem cells (ISCs) fuel the rapid turnover of the epithelium during homeostasis and might support epithelial regeneration after injury. In anatomically defined zones of the crypt stem cell niche, phenotypically distinct active and reserve ISC populations are believed to support homeostatic epithelial renewal and injury-induced regeneration, respectively. However, other cell types previously thought to be committed to differentiated states might also have ISC activity and participate in regeneration. Efforts are underway to reconcile the proposed relatively strict hierarchical relationships between reserve and active ISC pools and their differentiated progeny; findings from models provide evidence for phenotypic plasticity that is common among many if not all crypt-resident intestinal epithelial cells. We discuss the challenges to consensus on ISC nomenclature, technical considerations, and limitations inherent to methodologies used to define reserve ISCs, and the need for standardized metrics to quantify and compare the relative contributions of different epithelial cell types to homeostatic turnover and post-injury regeneration. Increasing our understanding of the high-resolution genetic and epigenetic mechanisms that regulate reserve ISC function and cell plasticity will help refine these models and could affect approaches to promote tissue regeneration after intestinal injury.

Integrated phosphorescence-based photonic biosensor (iPOB) for monitoring oxygen levels in 3D cell culture systems

Rivera KR, Pozdin VA, Young AT, Erb PD, Wisniewski NA, Magness ST, Daniele M

Biosens Bioelectron. 2019 Jan 1;123:131-140.  Epub 2018 Jul 21. PMID: 30060990

Abstract

Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.

Sox4 Promotes Atoh1-Independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates

Gracz AD, Samsa LA, Fordham MJ, Trotier DC, Zwarycz B, Lo YH, Bao K, Starmer J, Raab JR, Shroyer NF, Reinhardt RL, Magness ST

Gastroenterology. 2018 Nov;155(5):1508-1523. Epub 2018 Jul 25 PMID: 30055169

Abstract

BACKGROUND & AIMS: The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation. METHODS: We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation. RESULTS: Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1. CONCLUSIONS: We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection.

Organoid Cultures for Assessing Intestinal Epithelial Differentiation and Function in Response to Type-2 Inflammation

Zwarycz B, Gracz AD, Magness ST

Methods Mol Biol. 2018;1799:397-417. PMID: 29956167

Abstract

During helminth infection of the gastrointestinal tract, a complex Type-2 inflammatory response involving immunological and mucosal components is mounted to clear the infection and reestablish a physiologically normal state. This response is characterized by the secretion of key interleukins, which impact epithelial lineage allocation and drive tuft and goblet cell hyperplasia to lead to eventual clearance of parasitic organisms. While there have been advances toward understanding Type-2 inflammatory responses in the intestine, detailed cellular and molecular mechanisms of epithelial responses to general inflammation and specific inflammatory cytokines remain to be explored. Intestinal organoids represent a physiologically relevant in vitro model to study how Type-2 inflammation impacts stem cell maintenance and differentiation and offer a new approach for investigators to test compounds that modulate mechanisms involved in worm clearance. The methods described in this chapter include: (1) intestinal crypt and single cell isolation; (2) organoid culture and cytokine treatment, as well as methods for downstream organoid analyses; (3) gene expression analysis by qRT-PCR; (4) protein analysis by western blot, immunohistochemistry, and florescence-activated cell sorting; and (5) organoid self-renewal by serial passaging.

Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5- and Bmi1-Expressing Intestinal Cell Populations

Smith NR, Swain JR, Davies PS, Gallagher AC, Parappilly MS, Beach CZ, Streeter PR, Williamson IA, Magness ST, Wong MH

Cell Mol Gastroenterol Hepatol. 2018 Mar 10;6(1):79-96. eCollection 2018. PMID: 29928673

Abstract

BACKGROUND & AIMS: Continual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)+ intestinal stem cell (ISC) functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function. METHODS: We used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC-green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed. RESULTS: Two novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established. CONCLUSIONS: These data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.

Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche

Wang Y, Kim R, Hinman SS, Zwarycz B, Magness ST, Allbritton NL

Cell Mol Gastroenterol Hepatol. 2018 Jan 17;5(3):440-453. eCollection 2018 Mar. Review. PMID: 29675459

Abstract

The relationship between intestinal stem cells (ISCs) and the surrounding niche environment is complex and dynamic. Key factors localized at the base of the crypt are necessary to promote ISC self-renewal and proliferation, to ultimately provide a constant stream of differentiated cells to maintain the epithelial barrier. These factors diminish as epithelial cells divide, migrate away from the crypt base, differentiate into the postmitotic lineages, and end their life span in approximately 7 days when they are sloughed into the intestinal lumen. To facilitate the rapid and complex physiology of ISC-driven epithelial renewal, in vivo gradients of growth factors, extracellular matrix, bacterial products, gases, and stiffness are formed along the crypt-villus axis. New bioengineered tools and platforms are available to recapitulate various gradients and support the stereotypical cellular responses associated with these gradients. Many of these technologies have been paired with primary small intestinal and colonic epithelial cells to re-create select aspects of normal physiology or disease states. These biomimetic platforms are becoming increasingly sophisticated with the rapid discovery of new niche factors and gradients. These advancements are contributing to the development of high-fidelity tissue constructs for basic science applications, drug screening, and personalized medicine applications. Here, we discuss the direct and indirect evidence for many of the important gradients found in vivo and their successful application to date in bioengineered in vitro models, including organ-on-chip and microfluidic culture devices.

Cftr Modulates Wnt/β-Catenin Signaling and Stem Cell Proliferation in Murine Intestine

Strubberg AM, Liu J, Walker NM, Stefanski CD, MacLeod RJ, Magness ST, Clarke LL

Cell Mol Gastroenterol Hepatol. 2017 Dec 7;5(3):253-271. eCollection 2018 Mar. PMID: 29675451

Abstract

BACKGROUND & AIMS: Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/β-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/β-catenin signaling. METHODS: Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9 enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein-coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice.RESULTS:  Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/β-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition.CONCLUSIONS: CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins

Gunasekara DB, DiSalvo M, Wang Y, Nguyen DL, Reed MI, Speer J, Sims CE, Magness ST, Allbritton NL

Anal Chem. 2018 Feb 6;90(3):1941-1950. Epub 2018 Jan 12. PMID: 29281259

Abstract

Enterotoxins increase intestinal fluid secretion through modulation of ion channels as well as activation of the enteric nervous and immune systems. Colonic organoids, also known as colonoids, are functionally and phenotypically similar to in vivo colonic epithelium and have been used to study intestinal ion transport and subsequent water flux in physiology and disease models. In conventional cultures, organoids exist as spheroids embedded within a hydrogel patty of extracellular matrix, and they form at multiple depths, impairing efficient imaging necessary to capture data from statistically relevant sample sizes. To overcome these limitations, an analytical platform with colonic organoids localized to the planar surface of a hydrogel layer was developed. The arrays of densely packed colonoids (140 μm average diameter, 4 colonoids/mm2) were generated in a 96-well plate, enabling assay of the response of hundreds of organoids so that organoid subpopulations with distinct behaviors were identifiable. Organoid cell types, monolayer polarity, and growth were similar to those embedded in hydrogel. An automated imaging and analysis platform efficiently tracked over time swelling due to forskolin and fluid movement across the cell monolayer stimulated by cholera toxin. The platform was used to screen compounds associated with the enteric nervous and immune systems for their effect on fluid movement across epithelial cells. Prostaglandin E2 promoted increased water flux in a subset of organoids that resulted in organoid swelling, confirming a role for this inflammatory mediator in diarrheal conditions but also illustrating organoid differences in response to an identical stimulus. By allowing sampling of a large number of organoids, the arrayed organoid platform permits identification of organoid subpopulations intermixed within a larger group of nonresponding organoids. This technique will enable automated, large-scale screening of the impact of drugs, toxins, and other compounds on colonic physiology.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells

Wang Y, DiSalvo M, Gunasekara DB, Dutton J, Proctor A, Lebhar MS, Williamson IA, Speer J, Howard RL, Smiddy NM, Bultman SJ, Sims CE, Magness ST, Allbritton NL

Cell Mol Gastroenterol Hepatol. 2017 Mar 6;4(1):165-182. eCollection 2017 Jul. PMID: 29204504

Abstract

BACKGROUND & AIMS: Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished.

METHODS: The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified.

RESULTS: The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. CONCLUSIONS: This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.

Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells

Bhatt AP, Gunasekara DB, Speer J, Reed MI, Peña AN, Midkiff BR, Magness ST, Bultman SJ, Allbritton NL, Redinbo MR

ACS Infect Dis. 2018 Jan 12;4(1):46-52. Epub 2017 Nov 10. PMID: 29094594

Abstract

The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions.

In Vitro Generation of Mouse Colon Crypts

Wang Y, Gunasekara DB, Attayek PJ, Reed MI, DiSalvo M, Nguyen DL, Dutton JS, Lebhar MS, Bultman SJ, Sims CE, Magness ST, Allbritton NL

ACS Biomater Sci Eng. 2017 Oct 9;3(10):2502-2513. Epub 2017 Aug 29. PMID: 30854421

Abstract

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo has not been recapitulated in the organoid systems. We have used a microengineered platform with suitable extracellular matrix contacts and stiffness to generate a self-renewing mouse colonic epithelium that replicates key architectural and physiological functions found in vivo, including a surface lined with polarized crypts. Chemical gradients applied to the basal-luminal axis compartmentalized the stem/progenitor cells and promoted appropriate lineage differentiation along the in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model.

A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium

Wang Y, Gunasekara DB, Reed MI, DiSalvo M, Bultman SJ, Sims CE, Magness ST, Allbritton NL

Biomaterials. 2017 Jun;128:44-55. Epub 2017 Mar 6. PMID: 28288348

Abstract

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.

Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals

Azcarate-Peril MA, Ritter AJ, Savaiano D, Monteagudo-Mera A, Anderson C, Magness ST, Klaenhammer TR

Proc Natl Acad Sci U S A. 2017 Jan 17;114(3):E367-E375. Epub 2017 Jan 3. PMID: 28049818

Abstract

Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated "RP-G28," significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66). In this study, changes in the fecal microbiome were investigated using 16S rRNA amplicon pyrosequencing and high-throughput quantitative PCR. At day 36, bifidobacterial populations were increased in 27 of 30 of GOS subjects (90%), demonstrating a bifidogenic response in vivo. Relative abundance of lactose-fermenting Bifidobacterium, Faecalibacterium, and Lactobacillus were significantly increased in response to GOS. When dairy was introduced into the diet, lactose-fermenting Roseburia species increased from day 36 to day 66. The results indicated a definitive change in the fecal microbiome of lactose-intolerant individuals, increasing the abundance of lactose-metabolizing bacteria that were responsive to dietary adaptation to GOS. This change correlated with clinical outcomes of improved lactose tolerance.

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

Kechele DO, Blue RE, Zwarycz B, Espenschied ST, Mah AT, Siegel MB, Perou CM, Ding S, Magness ST, Lund PK, Caron KM

J Clin Invest. 2017 Feb 1;127(2):593-607.  Epub 2017 Jan 17. PMID: 28094771

Abstract

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation.

In Vitro Polarization of Colonoids to Create an Intestinal Stem Cell Compartment

Attayek PJ, Ahmad AA, Wang Y, Williamson I, Sims CE, Magness ST, Allbritton NL

PLoS One. 2016 Apr 21;11(4):e0153795.  PMID: 27100890

Abstract

The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.

SOX9 Maintains Reserve Stem Cells and Preserves Radio-resistance in Mouse Small Intestine.

 

Roche KC, Gracz AD, Liu XF, Newton V, Akiyama H, Magness ST

Gastroenterology. 2015 Nov;149(6):1553-1563. Epub 2015 Jul 11. PMID:26170137

Abstract

 

BACKGROUND & AIMS: Reserve intestinal stem cells (rISCs) are quiescent/slowly cycling under homeostatic conditions, allowing for their identification with label-retention assays. rISCs mediate epithelial regeneration after tissue damage by converting to actively proliferating stem cells (aISCs) that self renew and demonstrate multipotency, which are defining properties of stem cells. Little is known about the genetic mechanisms that regulate the production and maintenance of rISCs. High expression levels of the transcription factor Sox9 (Sox9(high)) are associated with rISCs. This study investigates the role of SOX9 in regulating the rISC state. METHODS: We used fluorescence-activated cell sorting to isolate cells defined as aISCs (Lgr5(high)) and rISCs (Sox9(high)) from Lgr5(EGFP) and Sox9(EGFP) reporter mice. Expression of additional markers associated with active and reserve ISCs were assessed in Lgr5(high) and Sox9(high) populations by single-cell gene expression analyses. We used label-retention assays to identify whether Sox9(high) cells were label-retatining cells (LRCs). Lineage-tracing experiments were performed in Sox9-CreERT2 mice to measure the stem cell capacities and radioresistance of Sox9-expressing cells. Conditional SOX9 knockout mice and inducible-conditional SOX9 knockout mice were used to determine whether SOX9 was required to maintain LRCs and rISC function. RESULTS:  Lgr5(high) and a subset of crypt-based Sox9(high) cells co-express markers of aISC and rISC (Lgr5, Bmi1, Lrig1, and Hopx). LRCs express high levels of Sox9 and are lost in SOX9-knockout mice. SOX9 is required for epithelial regeneration after high-dose irradiation. Crypts from SOX9-knockout mice have increased sensitivity to radiation, compared with control mice, which could not be attributed to impaired cell-cycle arrest or DNA repair. CONCLUSIONS: SOX9 limits proliferation in LRCs and imparts radiation resistance to rISCs in mice.

IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations.

Van Landeghem L, Santoro MA, Mah AT, Krebs AE, Dehmer JJ, McNaughton KK, Helmrath MA, Magness ST, Lund PK

FASEB J. 2015 Jul;29(7):2828-42.  Epub 2016 Jun 16.   PMID:25837582

Abstract

Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.

A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

 

Gracz AD, Williamson IA, Roche KC, Johnston MJ, Wang F, Wang Y, Attayek PJ, Balowski J, Liu XF, Laurenza RJ, Gaynor LT, Sims CE, Galenko JA, Li L, Allbritton NL, Magness ST.

Nat Cell Biol. 2015 Mar;17(3):340-9. Epub 2015 Feb 9 PMID: 25664616

Abstract

Stem cells reside in ‘niches’, where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell–niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a ‘microraft array’ (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell–niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

Defining Hierarchies of Stemness in the Intestine: Evidence from Biomarkers and Regulatoy Pathways

 

Gracz AD, Magness ST.

Am J Physiol Gastrointest Liver Physiol. 2014 Aug 1;307(3):G260-73. Epub 2014 Jun 12. Review  PMID:24924746

Abstract

For decades, the rapid proliferation and well-defined cellular lineages of the small intestinal epithelium have driven an interest in the biology of the intestinal stem cells (ISCs) and progenitors that produce the functional cells of the epithelium. Recent and significant advances in ISC biomarker discovery have established the small intestinal epithelium as a powerful model system for studying general paradigms in somatic stem cell biology, and facilitated elegant genetic and functional studies of stemness in the intestine. However, this newfound wealth of ISC biomarkers raises important questions of marker specificity. Furthermore, the ISC field must now begin to reconcile biomarker status with functional stemness, a challenge that is made more complex by emerging evidence that cellular hierarchies in the intestinal epithelium are more plastic than previously imagined, with some progenitor populations capable of dedifferentiating and functioning as ISCs following damage. In this review, we discuss the state of the ISC field in terms of biomarkers, tissue dynamics, and cellular hierarchies, and how these processes might be informed by earlier studies into signaling networks in the small intestine.

Impact of diet-induced obesity on intestinal stem cells: hyperproliferation but impaired intrinsic function that requires insulin/IGF1

 

Mah AT, Van Landeghem L, Gavin HE, Magness ST, Lund PK.

Endocrinology. 2014 Sep;155(9):3302-14. Epub 2014 Jun 10. PMID: 24914941

Abstract

It is well established that reduced nutrient intake decreases intestinal epithelial mass and crypt proliferation. Recent findings in model organisms and rodents indicate that these changes impact intestinal stem cells (ISC). In contrast, little is known about the impact of diet-induced obesity (DIO), a model of excess nutrient intake on ISC. We used a Sox9-EGFP reporter mouse to test the hypothesis that an adaptive response to DIO or associated hyperinsulinemia involves targeted expansion and hyperproliferation of ISC. The Sox9-EGFP reporter mouse allows study and isolation of ISC, and progenitors and differentiated lineages based on different Sox9-EGFP expression levels. Sox9-EGFP mice were fed high fat diet for 20 weeks to induce DIO and compared with littermate controls fed low fat rodent chow. Histology, fluorescence activated cell sorting (FACS) and mRNA analysis measured impact of DIO on jejunal crypt-villus morphometry, numbers and proliferation of different Sox9-EGFP cell populations and gene expression. An in vitro culture assay directly assessed functional capacity of isolated ISC. DIO mice exhibited significant increases in body weight and plasma glucose and insulin levels, indicative of reduced insulin sensitivity. DIO mice also displayed increases in circulating insulin-like growth factor 1 (IGF1) and intestinal Igf1 mRNA. DIO mice had increased villus height and crypt density but decreased intestinal length. DIO resulted in a selective expansion of Sox9-EGFPLow ISC and numbers of ISC in S-phase, and ISC expansion correlated with plasma insulin levels. In vitro, ISCs isolated from DIO mice formed fewer enteroids in standard 3D Matrigel culture compared to controls,indicative of impaired ISC function. This decrease in enteroid formation in ISCs from DIO mice was rescued when insulin, IGF1 or both were added. We conclude that DIO induces specific increases in ISC and ISC hyperproliferation in vivo, but acquired dependence on insulin or IGF1 for intrinsic survival and growth.

Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications

 

Ahmad AA, Wang Y, Gracz AD, Sims CE, Magness ST, Allbritton NL.

J Biol Eng. 2014 Apr 1;8:9.  eCollection 2014 PMID: 24690469

Abstract

BACKGROUND: New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising efficiency and reproducibility. Here murine crypt isolation yield and quality are optimized, and colonoid culture efficiency measured in microfabricated culture devices. RESULTS: An optimal incubation time of 60 min in a chelating buffer released 280,000 +/- 28,000 crypts from the stroma of a single colon with 79.3% remaining intact. Mechanical agitation using an average acceleration of 1.5 x g liberated the highest quality crypts with 86% possessing well-defined lumens. Culture in 50% Matrigel resulted in the highest colonoid formation efficiency of 33 +/- 5%. Immunostaining demonstrated that colonoids isolated under these conditions possessed stem/progenitor cells and differentiated cell lineages. Microfabrication substrates (glass, polystyrene, PDMS, and epoxy photoresists: SU-8 and 1002-F) were tested for compatibility with colonoid culture. PDMS promoted formation of 3-D colonoids containing stem/progenitor cells, while other substrates promoted outgrowth of a 2-D epithelial monolayer composed of differentiated cells. CONCLUSION: Improved crypt isolation and 3-D colonoid culture, along with an understanding of colonic epithelial cell behavior in the presence of microfabrication substrates will support development of 'organ-on-a-chip' approaches for studies using primary colonic epithelium.

In vitro generation of colonic epithelium from primary cells guided by microstructures

Wang Y, Ahmad AA, Sims CE, Magness ST, Allbritton NL.

Lab Chip. 2014 May 7;14(9):1622-31. Epub 2014 Mar 20. PMID: 24647645

Abstract

The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm, depth = 150 μm) followed by addition of Matrigel that formed micropockets locking the crypts within the microwells. Embedded crypts first underwent 3D expansion inside the wells. After the cells filled the microwells, they migrated onto the surrounding surface forming a 2D monolayer in the array regions without Matrigel. This unique 2D/3D hybrid culture generated a continuous, millimeter-scale colonic epithelial tissue in vitro, which resembled the polarized architecture (i.e. distinct proliferative and non-proliferative zones) and geometry of the colonic epithelium in vivo. This work initiates the construction of a "colon-on-a-chip" using primary cells/tissues with the ultimate goal of producing the physiologic structure and organ-level function of the colon.

Capture and 3D culture of colonic crypts and colonoids in a microarray platform

 

Wang Y, Ahmad AA, Shah PK, Sims CE, Magness ST, Allbritton NL.

Lab Chip. 2013 Dec 7;13(23):4625-34. PMID: 24113577

Abstract

The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm, depth = 150 μm) followed by addition of Matrigel that formed micropockets locking the crypts within the microwells. Embedded crypts first underwent 3D expansion inside the wells. After the cells filled the microwells, they migrated onto the surrounding surface forming a 2D monolayer in the array regions without Matrigel. This unique 2D/3D hybrid culture generated a continuous, millimeter-scale colonic epithelial tissue in vitro, which resembled the polarized architecture (i.e. distinct proliferative and non-proliferative zones) and geometry of the colonic epithelium in vivo. This work initiates the construction of a "colon-on-a-chip" using primary cells/tissues with the ultimate goal of producing the physiologic structure and organ-level function of the colon.

A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells

 

Magness ST, Puthoff BJ, Crissey MA, Dunn J, Henning SJ, Houchen C, Kaddis JS, Kuo CJ, Li L, Lynch J, Martin MG, May R, Niland JC, Olack B, Qian D, Stelzner M, Swain JR, Wang F, Wang J, Wang X, Yan K, Yu J, Wong MH

  

Am J Physiol Gastrointest Liver Physiol. 2013 Oct 15;305(8):G542-51.  Epub 2013 Aug 8 PMID: 3928185 

Abstract

Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.

Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay

 

Wang F, Scoville D, He XC, Mahe M, Box A, Perry J, Smith NR, Nanye NL, Davies PS, Fuller MK, Haug JS, McClain M, Gracz AD, Ding S, Stelzner M, Dunn JCY, Magness ST, Wong MH, Martin M, Helmrath M, and Li L.

Gastroenterology. 2013 Aug;145(2):383-95. Epub 2013 May 2. PMID: 23644405

Abstract

BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns, and difficulty to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. We used immunohistochemistry, real-time reverse transcription PCR, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein (GFP). We developed a culture protocol to facilitate the identification of functional ISCs from mice, and then tested the assay with human intestinal crypts and putative ISCs.

RESULTS: CD44+CD24loCD166+ cells, sorted by FACS from mouse small intestine and colon, expressed high levels of stem-cell associated genes. Transit amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78 lo/- putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25%-30% of single-sorted ISCs. CONCLUSIONS: We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers.CD44+CD24loCD166+, GRP78lo/- and c-Kit- facilitated identification of putative stem cells from the mouse small intestine and colon respectively. CD44+CD24-/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells

Gracz AD*, Fuller MK*, Wang F, Li L, Stelzner M, Dunn JCY, Martin M, Magness ST

Stem Cells. 2013 Sep;31(9):2024-30 PMID: 23553902

Abstract

Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive “active” stem cells as well as HOPX positive “facultative” stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 cells (CD24-/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease.

Restriction of intestinal stem cell expansion and the regenerative response by YAP

 

Barry ER, Morikawa T, Butler BL, Shrestha K, de la Rosa R, Yan KS, Fuchs CS, Magness ST, Smits R, Ogino S, Kuo CJ, Camargo FD.

Nature. 2013 Jan 3;493(7430):106-10. Epub 2012 Nov 25. PMID: 23178811

Abstract

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Mucosal healing and fibrosis after acute or chronic inflammation in wild type FVB-N mice and C57BL6 procollagen 1(I)-promoter-GFP reporter mice.

Ding S, Walton KLW, Blue RE, MacNaughton K, Magness ST, Lund PK

PLoS One. 2010 Aug 16;5(8):e12191. PMID: 22880035 

Abstract

Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn's disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models. Methods: FVB-N wild type mice and C57BL6 procollagen α1(I)-GFP reporter mice were given one (DSS1) or two (DSS2) cycles of 3% DSS (5 days/cycle) followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3 and DSS2+7. Procollagen α1(I)-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA) or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I) mRNA. Results: Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I)-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin+ cells and much fewer α-SMA+ cells. GFP mRNA strongly correlated with collagen α1(I) mRNA. Conclusions: One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I)-GFP

transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic experimental intestinal inflammation and fibrosis.

Identification, isolation, and culture of intestinal epithelial stem cells from murine intestine

 

Gracz AD, Puthoff BJ, Magness ST.

Methods Mol Biol. 2012;879:89-107 PMID: 22610555

Abstract

The study of adult stem cell populations provides insight into the mechanisms that regulate tissue maintenance in normal physiology and many disease states. With an impressive rate of epithelial renewal driven by a pool of multipotent stem cells, the intestine is a particularly advantageous model system for the study of adult stem cells. Until recently, the isolation and in vitro study of intestinal epithelial stem cells (IESCs) was not possible due to the lack of biomarkers and culture techniques. However, advances in molecular characterization and culture of IESCs have made in vitro studies on this cell type amenable to most laboratories. The methods described in this chapter will allow the investigator to adapt newly established techniques toward downstream analysis of IESCs in vitro.

A nomenclature for intesintal in vitro cultures

 

Stelzner M, Helmrath MA, Dunn JC, Henning SJ, Houchen CW, Kuo CJ, Lynch JP, Li L, Magness ST, Martin MG, Wong MH, Yu J.

Am J Physiol Gastrointest Liver Physiol. 2012 Jun 15;302(12):G1359-63. Epub 2012 Mar 29. Review. PMID: 22461030

Abstract

Many advances have been reported in the long-term culture of intestinal mucosal cells in recent years. A significant number of publications have described new culture media, cell formations, and growth patterns. Furthermore, it is now possible to study, e.g., the capabilities of isolated stem cells or the interactions between stem cells and mesenchyme. However, at the moment there is significant variation in the way these structures are described and named. A standardized nomenclature would benefit the ability to communicate and compare findings from different laboratories using the different culture systems. To address this issue, members of the NIH Intestinal Stem Cell Consortium herein propose a systematic nomenclature for in vitro cultures of the small and large intestine. We begin by describing the structures that are generated by preparative steps. We then define and describe structures produced in vitro, specifically: enterosphere, enteroid, reconstituted intestinal organoid, induced intestinal organoid, colonosphere, colonoid, and colonic organoid.

Activation of two distinct Sox9-EGFP expressing intestinal stem cell populations during crypt regeneration after irradiation

 

Van Landeghem, L.M., Santoro, A., Krebs, A., Mah, A.T., Dehmer, J.J., Gracz., A.D., Scull, B.P., McNaughton, K., Magness, S.T., Lund, P.K.,

Am J Physiol Gastrointest Liver Physiol. 2012 May 15;302(10):G1111-32. Epub 2012 Feb 23. PMID: 22361729

Abstract

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

Mitochondrail DNA polymerase editing mutation, POLGD257A, disturbes stem-progenitor cell cycling in the small intestine and restricts excess fat absorption

 

Fox, R.G., Magness, S.T., Kujoth, G.C., Prolla, T.A., Maeda, N

Am J Physiol Gastrointest Liver Physiol. 2012 May 1;302(9):G914-24. Epub 2012 Feb 16. PMID: 22345551

Abstract

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

Notch Signaling Modulates Proliferation and Differentiation of Intestinal Crypt Base Columnar Stem Cells. Development

 

VanDussen, K.L., Carulli, A.J., Keeley, T.M., Patel, S.R., Puthoff, B.J., Magness, S.T., Tran, I.T., Maillard, I., Siebel, C.W., Kolterud, A., Grosse, A.S., Gumucio, D.L., Ernst, S.A., Tsai, Y-H, Dempsey, P.J., and Samuelson, L.C.

Development. 2012 Feb;139(3):488-97.  Epub 2011 Dec 21. PMID: 2190634

Abstract

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-J binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

Distinct levels of Sox9 expression mark colon epithelial stem cells that form colonoids in culture

 

Ramalingam, H., Daughtridge, G.W., Johnston, M.J., Gracz, A.D. Magness, S.T.

Am J Physiol Gastrointest Liver Physiol. 2012 Jan 1;302(1):G10-20. Epub 2011 Oct 13. PMID: 21995959

Abstract

Sox9 is an high-mobility group box transcription factor that is expressed in the stem cell zone of the small intestine and colon. We have previously used a Sox9EGFP mouse model to demonstrate that discrete levels of Sox9 expression mark small intestine epithelial stem cells that form crypt/villus-like structures in a three-dimensional culture system. In the present study, we hypothesized that discrete levels of Sox9 expression would also mark colonic epithelial stem cells (CESCs). Using the Sox9EGFP mouse model, we show that lower levels of Sox9 mark cells in the transit-amplifying progenitor cell zone, while higher levels of Sox9 mark cells in the colonic crypt base. Furthermore, we demonstrate that variable SOX9 levels persist in cells of colonic adenomas from mice and humans. Cells expressing lower Sox9 levels demonstrate gene expression profiles consistent with more differentiated populations, and cells expressing higher Sox9 levels are consistent with less differentiated populations. When placed in culture, cells expressing the highest levels of Sox9 formed "colonoids," which are defined as bodies of cultured colonic epithelial cells that possess multiple cryptlike structures and a pseudolumen. Cells expressing the highest levels of Sox9 also demonstrate multipotency and self-renewal in vitro, indicating functional stemness. These data suggest a dose-dependent role for Sox9 in normal CESCs and cells comprising colon tumors. Furthermore, distinct Sox9 levels represent a new biomarker to study CESC and progenitor biology in physiological and disease states.

Sry-box (SOX) transcription factors in gastrointestinal physiology and disease

 

Gracz, A.D., Magness S.T.

Am J Physiol Gastrointest Liver Physiol. 2011 Apr;300(4):G503-15. Epub 2011 Feb 3. Review. PMID: 21995959

Abstract

The genetic mechanisms underlying tissue maintenance of the gastrointestinal tract are critical for the proper function of the digestive system under normal physiological stress. The identification of transcription factors and related signal transduction pathways that regulate stem cell maintenance and lineage allocation is attractive from a clinical standpoint in that it may provide targets for novel cell- or drug-based therapies. Sox [sex-determining region Y (Sry) box-containing] factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems and might provide valuable insight toward the understanding of these processes in endodermally derived tissues of the gastrointestinal tract. In this review, we focus on the known genetic functions of Sox factors and their roles in epithelial tissues of the esophagus, stomach, intestine, colon, pancreas, and liver. Additionally, we discuss pathological conditions in the gastrointestinal tract that are associated with a dysregulation of Sox factors. Further study of Sox factors and their role in gastrointestinal physiology and pathophysiology may lead to advances that facilitate control of tissue maintenance and development of advanced clinical therapies.

Sorting mouse jejunal epithelial cells with CD24 yeilds a population with characteristics of intestinal epithelial stem cells

 

von Furstenberg R.J., Gulati A.S., Baxi A., Doherty J.M., Stappenbeck T.S., Gracz A.D., Magness S.T., Henning S.J.

Am J Physiol Gastrointest Liver Physiol. 2011 Mar;300(3):G409-17. Epub 2010 Dec 23. PMID: 21183658

Abstract

Intestinal stem cells (ISCs) have been studied for more than three decades; however, their isolation has remained a challenge. We hypothesized that, just as for stem cells of other tissues, one or more membrane markers would allow positive selection of ISCs by antibody-based sorting. To explore this hypothesis, microarray data of putative ISC fractions generated by side population sorting and laser capture microdissection were subjected to bioinformatic analysis to identify common membrane antigens. The microarray comparison suggested CD24 as a candidate surface marker, and immunohistochemistry showed expression of CD24 in epithelial cells of crypt bases. Flow cytometry of jejunal epithelial preparations revealed a CD24(+) CD45(-) fraction comprising 1% of the cells. Analysis with epithelial cell adhesion molecule and CD31 confirmed that the cell preparations were epithelial and without endothelial contamination. Cycling cells identified by prior injection with 5-ethynyl-2'-deoxyuridine were found predominantly in the CD24(lo) subfraction. Transcript analysis by real-time RT-PCR showed this subfraction to be enriched in the ISC markers leucine-rich-repeat-containing G-protein-coupled receptor 5 (40-fold) and Bmi1 (5-fold), but also enriched in lysozyme (10-fold). Flow cytometry with anti-lysozyme antibodies demonstrated that Paneth cells comprise 30% of the CD24(lo) subfraction. Additional flow analyses with leucine-rich-repeat-containing G-protein-coupled receptor 5-enhanced green fluorescent protein (EGFP) epithelium demonstrated colocalization of EGFP(hi) and CD24(lo). In contrast, CD24 cells were negative for the quiescent ISC marker doublecortin and CaM kinase-like-1. Culture of CD24(lo) cells in Matrigel generated organoid structures, which included all four epithelial lineages, thus giving functional evidence for the presence of ISCs. We conclude that the CD24(lo) fraction of jejunal epithelium is highly enriched with cycling ISCs. This isolation method should be useful to many investigators in the field to advance both the basic understanding of ISC biology and the therapeutic applications of ISCs.

High-fat diet: bacteria interactions promote intestinal inflammation which precedes and correlates with obesity and insulin resistance in mouse

Ding S, Chi MM, Scull BP, Rigby R, Schwerbrock NM, Magness ST, Jobin C, Lund PK

PLoS One. 2010 Aug 16;5(8):e12191. PMID: 20808947

Abstract

BACKGROUND: Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2-16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-alpha mRNA and activation of a NF-kappaB(EGFP) reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-alpha mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-kappaB(EGFP) in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-kappaB(EGFP) in GF NF-kappaB(EGFP) mice. CONCLUSIONS/SIGNIFICANCE: Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance.

Sox9-expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro

 

Gracz, A.D., Ramalingam, S., Magness S.T.

Am J Physiol Gastrointest Liver Physiol. 2010 May;298(5):G590-600. Epub 2010 Feb 25. PMID: 20185687

Abstract

The inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a Sox9(EGFP) mouse model, we demonstrate that Sox9(EGFP) fluorescence signatures can be used to differentiate between and enrich for progenitors (Sox9(EGFPsubLo)) and multipotent IESCs (Sox9(EGFPlo)). Sox9(EGFPlo) cells generate "organoids" in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of Sox9(EGFPlo) cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the Sox9(EGFPlo) population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model.

Insulin receptor substrate-1 deficiency promotes apoptosis in the putative intestinal crypt stem cell region, limits Apcmin/+ tumors, and regulates Sox9

 

Ramocki, N.M, Wilkins, H.R., Magness S.T., Simmons, J.G., Scull, B.P., McNaughton, K.K., Lund, P.K.

Endocrinology. 2008 Jan;149(1):261-7. Epub 2007 Oct 4. PMID: 17916629

Abstract

Reduced apoptosis of crypt stem/progenitor cells and elevated insulin and IGFs are linked to colon cancer risk. Insulin receptor substrate-1 (IRS-1) mediates the actions of insulin, IGF-I, and IGF-II, but the role of endogenous IRS-1 in crypt apoptosis and cancer is undefined. Using IRS-1(-/-), IRS-1(+/-), and IRS-1(+/+) mice, we tested the hypothesis that reduced IRS-1 expression increases apoptosis of intestinal crypt cells and protects against Apc(min/+) (Min)/beta-catenin-driven intestinal tumors. Expression of Sox9, a transcriptional target of Tcf/beta-catenin and putative biomarker of crypt stem cells, was assessed in intestine of different IRS-1 genotypes and cell lines. Irradiation-induced apoptosis was significantly increased in the crypts and crypt stem cell region of IRS-1-deficient mice. Tumor load was significantly reduced by 31.2 +/- 14.6% in IRS-1(+/-)/Min and by 64.1 +/- 7.6% in IRS-1(-/-)/Min mice, with more prominent reductions in tumor number than size. Compared with IRS-1(+/+)/Min, IRS-1(-/-)/Min mice had fewer Sox9-positive cells in intestinal crypts and reduced Sox9 mRNA in intestine. IRS-1 overexpression increased Sox9 expression in an intestinal epithelial cell line. We conclude that even small reductions in endogenous IRS-1 increase apoptosis of crypt stem or progenitor cells, protect against beta-catenin-driven intestinal tumors, and reduce Sox9, a Tcf/beta-catenin target and putative stem/progenitor cell biomarker.

Distinct SOX9 Levels Differentially Mark Stem/Progenitor Populations and Enteroendocrine Cells of the Small Intestine Epithelium

Formeister E.J., Sionas A.L., Lorance D.K., Barkley C.L., Lee G.H., Magness S.T.

Am J Physiol Gastrointest Liver Physiol. 2009, May; 296(5):G1108-18. Epub 2009 Feb 19 PMID: 19228882

Abstract

SOX transcription factors have the capacity to modulate stem/progenitor cell proliferation and differentiation in a dose-dependent manner. SOX9 is expressed in the small intestine epithelial stem cell zone. Therefore, we hypothesized that differential levels of SOX9 may exist, influencing proliferation and/or differentiation of the small intestine epithelium. Sox9 expression levels in the small intestine were investigated using a Sox9 enhanced green fluorescent protein (Sox9(EGFP)) transgenic mouse. Sox9(EGFP) levels correlate with endogenous SOX9 levels, which are expressed at two steady-state levels, termed Sox9(EGFPLO) and Sox9(EGFPHI). Crypt-based columnar cells are Sox9(EGFPLO) and demonstrate enriched expression of the stem cell marker, Lgr5. Sox9(EGFPHI) cells express chromogranin A and substance P but do not express Ki67 and neurogenin3, indicating that Sox9(EGFPHI) cells are postmitotic enteroendocrine cells. Overexpression of SOX9 in a crypt cell line stopped proliferation and induced morphological changes. These data support a bimodal role for SOX9 in the intestinal epithelium, where low SOX9 expression supports proliferative capacity, and high SOX9 expression suppresses proliferation.