@article{sheahan_theriot_cortes_dekaney_2022, title={Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells}, volume={14}, ISSN={["1949-0984"]}, DOI={10.1080/19490976.2021.2018898}, abstractNote={ABSTRACT Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.}, number={1}, journal={GUT MICROBES}, author={Sheahan, Breanna J. and Theriot, Casey M. and Cortes, Jocsa E. and Dekaney, Christopher M.}, year={2022}, month={Dec} }
 @article{sheahan_freeman_keeley_samuelson_roper_hasapis_lee_dekaney_2021, title={Epithelial Regeneration After Doxorubicin Arises Primarily From Early Progeny of Active Intestinal Stem Cells}, volume={12}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2021.01.015}, abstractNote={<h3>Background & Aims</h3> aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. <h3>Methods</h3> We monitored DXR's effect on aISCs by enumerating Lgr5-eGFP<sup>+</sup> and Olfm4<sup>+</sup> crypts, cleaved caspase-3 (CASP3<sup>+</sup>) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (<i>Bmi1</i><sup>+</sup>, <i>Hopx</i><sup>+</sup>, <i>Dll1</i><sup>+</sup>, and <i>Defa6</i><sup>+</sup>) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. <h3>Results</h3> Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3<sup>+</sup> cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3<sup>+</sup> cells). There was minimal regenerative contribution from <i>Bmi1</i><sup>+</sup>, <i>Hopx</i><sup>+</sup>, <i>Dll1</i><sup>+</sup>, and <i>Defa6</i><sup>+</sup>-expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU<sup>+</sup>, and expressed low levels of DDR transcripts. <h3>Conclusions</h3> We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs' robust DDR, which facilitates expeditious apoptosis.}, number={1}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Sheahan, Breanna J. and Freeman, Ally N. and Keeley, Theresa M. and Samuelson, Linda C. and Roper, Jatin and Hasapis, Stephanie and Lee, Chang-Lung and Dekaney, Christopher M.}, year={2021}, pages={119–140} }
 @article{dekaney_2021, title={Hyperproliferation of the Intestinal Epithelium}, volume={12}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2021.06.010}, abstractNote={Intestinal epithelial stem cells and transit amplifying cells residing within the intestinal crypts exist in an exquisitely regulated environment, which tightly controls their ability to proliferate and differentiate. During development, adult homeostasis, and repair, cells of the stem cell niche produce a cadre of growth factors, including epidermal growth factor (EGF), that drive proliferation and stem cell renewal. Classically, we use chemical inhibitors and/or genetic knockouts to investigate the roles these factors play by signaling through their cognate receptors. However, overexpression or activating mutations of receptors, including members of the EGF receptor family, which can disrupt normal functional regulation, could provide useful insight into how growth factor signaling affects stem cell dynamics. In this issue of Cellular and Molecular Gastroenterology and Hepatology, Murray et al1Murray E. Cheng X. Krishna A. Jin X. Ohara T.E. Stappenbeck T.S. Bose R. HER2 and APC mutations promote altered crypt-villus morphology and marked hyperplasia in the intestinal epithelium.Cell Mol Gastroenterol Hepatol. 2021; 12: 1105-1120Abstract Full Text Full Text PDF Scopus (3) Google Scholar explore how an activating mutation in HER2, a member of the EGF receptor family, impacts small intestinal and colonic architecture, expression of stem cell genes, lineage allocation, and stem cell functionality via enteroid and colonoid cultures. To accomplish this, the authors generated a transgenic mouse that expressed HER2V777L, an activating mutation commonly found in colon cancer, under the control of the ROSA26 promoter and crossed it with the Lgr5-eGFP-IRES-CreER mouse line to induce expression of constitutively activated HER2 in intestinal epithelial cells. Interestingly, epithelial expression of HER2V777L elicited disparate responses in the small intestine and colon. In the small intestine, the authors found that epithelial expression of HER2V777L increased crypt depth, villus height, and proliferation, which occurred concomitantly with increased numbers of OLFM4+ cells at the crypt base. Furthermore, they found a general increase in mRNA expression of stem cell markers and Wnt target genes. Those changes were accompanied by increased budding of enteroids from transgenic compared with control mice. In contrast, although HER2V777L expression increased crypt depth and proliferation in colonic epithelium, these increases were associated with a general decrease in mRNA expression of stem cell-related and Wnt target genes. In addition, unlike small intestinal enteroids, colon organoids from transgenic mice did not show any morphologic differences. These data highlight the need for further comparative investigations of epithelial stem cell niches along the length of the gastrointestinal tract. Expansion of OLFM4+ cells in the small intestine came at the expense of Paneth cell numbers. Although overall mRNA expression of secretory cell markers was elevated in transgenic mice, further analysis revealed a significant increase in the number of lysozyme-positive cells within the villus epithelium. Costaining with MMP7 (a Paneth cell marker) and Muc2 (a goblet cell marker) confirmed that these cells were intermediate cells. These data raise interesting questions about the role EGF plays in lineage allocation and what controls Paneth cell maturation, and point to the need for cell-specific evaluation of expression of activating mutations, such as HER2V777L. Finally, to examine the role of co-occurring mutations on tumorigenesis the authors crossed their HER2V777L mouse line with the APCmin mouse line producing a model of 2 of the most prevalent mutations found in colon cancer. To their surprise, the authors did not find any significant increase in the number of Ki-67-positive cells per adenoma, average adenoma size, or number per mouse. However, they did find hyperproliferative regions characterized by HER2V777L-positive, proliferative crypts that protruded toward the villus tips. It should be noted that these tissues were evaluated only 10 days after the final tamoxifen treatment, which points to the rapidity with which these double mutations can affect normal crypt dynamics. The findings presented in this study bring to light the premise that intestinal epithelial cells of the crypt, and in particular, intestinal stem cells, can differ in their response to growth factors and other trophic factors in the small intestine and colon, and may differ along the length of the gastrointestinal tract. Future comparative studies could help delineate differences and similarities between stem cell niches within different intestinal regions and potentially provide better understanding of how these regions deal with disease-associated gene mutations. HER2 and APC Mutations Promote Altered Crypt-Villus Morphology and Marked Hyperplasia in the Intestinal EpitheliumCellular and Molecular Gastroenterology and HepatologyVol. 12Issue 3PreviewThe Cancer Genome Atlas (TCGA) project has identified HER2 mutations or amplification in 7% of colon cancers. In addition to HER2 mutations, colon cancer patients also possess co-occurring mutations in genes such as APC. Here, we investigated the role of HER2 and APC mutations on the crypt-villus architecture of the intestinal epithelium, localization of secretory cells, and expression of intestinal stem cell markers. Full-Text PDF Open Access}, number={3}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Dekaney, Christopher M.}, year={2021}, pages={1145–1146} }
 @article{shanahan_kanke_oyesola_hung_koch-laskowski_singh_peck_biraud_sheahan_cortes_et al._2021, title={Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types}, volume={321}, ISSN={["1522-1547"]}, DOI={10.1152/ajpgi.00222.2021}, abstractNote={MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely co-transcribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most-enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.}, number={6}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY}, author={Shanahan, Michael T. and Kanke, Matt and Oyesola, Oyebola O. and Hung, Yu-Han and Koch-Laskowski, Kieran and Singh, Ajeet P. and Peck, Bailey C. E. and Biraud, Mandy and Sheahan, Breanna and Cortes, Josca E. and et al.}, year={2021}, month={Dec}, pages={G668–G681} }
 @misc{cray_sheahan_dekaney_2021, title={Secretory Sorcery: Paneth Cell Control of Intestinal Repair and Homeostasis}, volume={12}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2021.06.006}, abstractNote={Paneth cells are professional secretory cells that classically play a role in the innate immune system by secreting antimicrobial factors into the lumen to control enteric bacteria. In this role, Paneth cells are able to sense cues from luminal bacteria and respond by changing production of these factors to protect the epithelial barrier. Paneth cells rely on autophagy to regulate their secretory capability and capacity. Disruption of this pathway through mutation of genes, such as Atg16L1, results in decreased Paneth cell function, dysregulated enteric microbiota, decreased barrier integrity, and increased risk of diseases such as Crohn's disease in humans. Upon differentiation Paneth cells migrate downward and intercalate among active intestinal stem cells at the base of small intestinal crypts. This localization puts them in a unique position to interact with active intestinal stem cells, and recent work shows that Paneth cells play a critical role in influencing the intestinal stem cell niche. This review discusses the numerous ways Paneth cells can influence intestinal stem cells and their niche. We also highlight the ways in which Paneth cells can alter cells and other organ systems.}, number={4}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Cray, Paul and Sheahan, Breanna J. and Dekaney, Christopher M.}, year={2021}, pages={1239–1250} }
 @article{cray_sheahan_cortes_dekaney_2020, title={Doxorubicin increases permeability of murine small intestinal epithelium and cultured T84 monolayers}, volume={10}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-020-78473-1}, abstractNote={Abstract Enteric bacteria and/or their products are necessary for doxorubicin (DXR)-induced small intestine mucosal damage. While DXR does not induce gross loss of epithelium, others have shown elevated serum endotoxin after DXR administration. However, the mechanism of movement is unknown. We hypothesized that DXR treatment resulted in increased paracellular translocation of bacteria or bacterial products through the small intestinal epithelium. We measured permeability after DXR administration using transepithelial resistance and macromolecular flux and assessed tight junctional gene expression and protein localization both in vitro using T84 cells and ex vivo using murine jejunum. DXR treatment increased flux of 4 kDa dextrans in mouse jejenum, but increased flux of 4, 10 and 20 kDa dextrans in T84 cells. Following DXR, we observed increased permeability, both in vitro and ex vivo, independent of bacteria. DXR induced increased expression of Cldn2 and Cldn4 in murine small intestine but increased only CLDN2 expression in T84 cells. DXR treatment induced disorganization of tight junctional proteins. We conclude that DXR increases paracellular transit of small macromolecules, including bacterial products, through the epithelium, by altering expression of tight junctional components and dynamic loosening of cellular tight junctions.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Cray, Paul and Sheahan, Breanna J. and Cortes, Jocsa E. and Dekaney, Christopher M.}, year={2020}, month={Dec} }
 @article{singh_hung_shanahan_kanke_bonfini_dame_biraud_peck_oyesola_freund_et al._2020, title={Enteroendocrine Progenitor Cell-Enriched mir-7 Regulates Intestinal Epithelial Proliferation in an Xiap-Dependent Manner}, volume={9}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2019.11.001}, abstractNote={The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis.We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice.First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling.This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.}, number={3}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Singh, Ajeet P. and Hung, Yu-Han and Shanahan, Michael T. and Kanke, Matt and Bonfini, Alessandro and Dame, Michael K. and Biraud, Mandy and Peck, Bailey C. E. and Oyesola, Oyebola O. and Freund, John M. and et al.}, year={2020}, pages={447–464} }
 @article{jones_brindley_elder_myers_rajala_dekaney_mcnamee_frey_shroyer_dempsey_2019, title={Cellular Plasticity of Defa4(Cre)-Expressing Paneth Cells in Response to Notch Activation and Intestinal Injury}, volume={7}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2018.11.004}, abstractNote={Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury.Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo.Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner.Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.}, number={3}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Jones, Jennifer C. and Brindley, Constance D. and Elder, Nicholas H. and Myers, Martin G., Jr. and Rajala, Michael W. and Dekaney, Christopher M. and McNamee, Eoin N. and Frey, Mark R. and Shroyer, Noah F. and Dempsey, Peter J.}, year={2019}, pages={533–554} }
 @article{dekaney_king_sheahan_cortes_2019, title={Mist1 Expression Is Required for Paneth Cell Maturation}, volume={8}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2019.07.003}, abstractNote={BackgroundPaneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells.MethodsMist1+/+ and Mist1–/– mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function.ResultsAbsence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1–/– mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo.ConclusionsMIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation. Paneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells. Mist1+/+ and Mist1–/– mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function. Absence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1–/– mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo. MIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.}, number={4}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Dekaney, Christopher M. and King, Stephanie and Sheahan, Breanna and Cortes, Jocsa E.}, year={2019}, pages={549–560} }
 @article{sheahan_dekaney_2018, title={Decrypting the Crypt: Novel Monoclonal Antibodies to Identify Intestinal Stem Cell Populations}, volume={6}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2018.04.006}, abstractNote={Despite extensive efforts to fully characterize and identify the stem cell pools at the base of the small intestinal crypts, there remains controversy over the precise identity of these cells. The active crypt base columnar cell, labeled by leucine-rich G-protein–coupled receptor 5 (Lgr5), is the workhorse of epithelial renewal, but other cell populations are able to contribute to this renewal, particularly after intestinal damage. The accurate labeling of these additional stem cell populations is plagued by the small numbers of these cells and the plasticity of the cell itself. Even labeling of the active stem cell population by Lgr5-enhanced green fluorescent protein (EGFP) has been imperfect, with mosaic silencing in the commonly used Lgr5EGFP-internal ribosome entry site (IRES)-Cre-estrogen receptor binding domain fusion (IRES-Cre-ER) transgenic mouse, and concurrent expression of markers of other stem cell populations observed in these active intestinal stem cells (ISCs). In addition, the use of transgenes to label intestinal stem cells cannot be applied to human beings and therefore direct application of findings in these models can be difficult. In the recent publication by Smith et al1Smith N.R. Swain J.R. Davies P.S. Gallagher A.C. Parappilly M.S. Beach C.Z. Streeter P.R. Williamson I.A. Magness S.T. Wong M.H. Monoclonal antibodies reveal dynamic plasticity between Lgr5- and Bmi1-expressing intestinal cell populations.Cell Mol Gastroenterol Hepatol. 2018; 6: 79-96Abstract Full Text Full Text PDF Scopus (13) Google Scholar the researchers confronted this problem by raising antibodies to various intestinal epithelial populations, then screening these for immunoreactivity to identified cells using a variety of methods. The investigators were successfully able to prove, via single-cell growth analyses, immunofluorescence, and gene expression, that the monoclonal antibody (mAb) F5C12 marks a population with essentially identical properties to Lgr5GFP sorted cells. Notably, the F5C12 mAb does not mark slow-cycling crypt cells or villous epithelium. A second mAb, E5D10, was highly expressed on villous epithelium, with low expression present in the crypt. Therefore, E5D10lo/F5C12- sorted cells contain all other crypt cells that are not actively cycling ISCs. Although Smith et al1Smith N.R. Swain J.R. Davies P.S. Gallagher A.C. Parappilly M.S. Beach C.Z. Streeter P.R. Williamson I.A. Magness S.T. Wong M.H. Monoclonal antibodies reveal dynamic plasticity between Lgr5- and Bmi1-expressing intestinal cell populations.Cell Mol Gastroenterol Hepatol. 2018; 6: 79-96Abstract Full Text Full Text PDF Scopus (13) Google Scholar showed that this population included Bmi1GFP cells with the ability to form spheroids in culture, they did comment that it also included Paneth cells, as indicated by higher lysozyme messenger RNA expression. It would have been interesting to further discriminate the E5D10lo/F5C12- population by ulex europaeus agglutinin (UEA)+ or side-scattered light (SSC)hi to remove the influence of concurrently isolated Paneth cells. However, because the Bmi1-expressing cell population has been reported to be insensitive to Wnt signaling, removal of the epithelial source of Wnt is unlikely to have altered the conclusions of the study. This also was supported by the differential response of the subsequent ex vivo cultures to the lack of R-spondin1, in which enteroids produced by actively cycling cells (F5C12+) were unable to survive the loss of Wnt enhancement, but the E5D10lo/F5C12- spheroids continued to proliferate. Bmi1GFP cells have been identified previously as committed enteroendocrine precursors that retain the ability to dedifferentiate into an active stem cell. This functionality likely relies on a facile alteration of their chromatin signature back to an active ISC profile. Disappointingly, Bmi1Cre-estrogen receptor binding domain fusion (IRES-Cre-ER); tandem dimeric tomato marks more cells than just those marked by Bmi1GFP, which makes lineage tracing from these cells difficult to reconcile with the signatures of the Bmi1GFP-expressing cells. This study used Bmi1CreERT2;tdTom only in the ex vivo cultures of E5D10lo/F5C12- flow sorted cells, which the investigators had identified previously as broadly expressing Bmi1GFP. Therefore, the Bmi1CreERT2;tdTom cells, when isolated by E5D10lo/F5C12-, likely represent the overlap in populations identified by the Bmi1GFP and Bmi1CreERT2;tdTom transgenes. As such, this E5D10lo/F5C12- population represents cells possessing the ability to restore stem cell function ex vivo. By using these novel mAbs to isolate different populations for ex vivo culture, the investigators showed a temporal plasticity in ISC gene expression. That is, isolated actively cycling ISCs (F5C12+) did not express Lgr5, as indicated by messenger RNA and by loss of Lgr5GFP expression, from 2 to 5 days after placement into culture. In addition, lineage tracing of Lgr5GFP-IRES-CreER cells, when induced with tamoxifen at day 2, showed minimal lineage tracing events. Instead, these ex vivo cultures had increased levels of Bmi1 transcripts on day 2. The investigators speculate that, when grown from isolated single cells, actively cycling stem cells transiently express a transcript profile consistent with slow-cycling stem cells. This coincides with the phenotypic appearance of these F5C12+ cultures: more spheroid than enteroid. Although this finding provides an exciting insight into the plasticity of these stem cell populations, it remains to be seen whether this holds true in vivo. Finally, if the F5C12 mAb continued to mark these cells as they transition through a slow-cycling-like phase, then the true utility of this mAb lies in the ability to follow active intestinal stem cells through damage and repair phases. This article represents an exciting and novel approach to isolation and identification of separate pools of intestinal stem cells. Furthermore, it describes potentially valuable tools for use in studies using transgenic and nontransgenic mice, large animal models, and human beings. Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5- and Bmi1-Expressing Intestinal Cell PopulationsCellular and Molecular Gastroenterology and HepatologyVol. 6Issue 1PreviewContinual 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. Full-Text PDF Open Access}, number={1}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Sheahan, Breanna and Dekaney, Christopher M.}, year={2018}, pages={121–122} }
 @article{rigby_carr_orgel_king_lund_dekaney_2016, title={Intestinal bacteria are necessary for doxorubicin-induced intestinal damage but not for doxorubicin-induced apoptosis}, volume={7}, ISSN={1949-0976 1949-0984}, url={http://dx.doi.org/10.1080/19490976.2016.1215806}, DOI={10.1080/19490976.2016.1215806}, abstractNote={ABSTRACT Doxorubicin (DOXO) induces significant, but transient, increases in apoptosis in the stem cell zone of the jejunum, followed by mucosal damage involving a decrease in crypt proliferation, crypt number, and villus height. The gastrointestinal tract is home to a vast population of commensal bacteria and numerous studies have demonstrated a symbiotic relationship between intestinal bacteria and intestinal epithelial cells (IEC) in maintaining homeostatic functions of the intestine. However, whether enteric bacteria play a role in DOXO-induced damage is not well understood. We hypothesized that enteric bacteria are necessary for induction of apoptosis and damage associated with DOXO treatment. Conventionally raised (CONV) and germ free (GF) mice were given a single injection of DOXO, and intestinal tissue was collected at 6, 72, and 120 h after treatment and from no treatment (0 h) controls. Histology and morphometric analyses quantified apoptosis, mitosis, crypt depth, villus height, and crypt density. Immunostaining for muc2 and lysozyme evaluated Paneth cells, goblet cells or dual stained intermediate cells. DOXO administration induced significant increases in apoptosis in jejunal epithelium regardless of the presence of enteric bacteria; however, the resulting injury, as demonstrated by statistically significant changes in crypt depth, crypt number, and proliferative cell number, was dependent upon the presence of enteric bacteria. Furthermore, we observed expansion of Paneth and goblet cells and presence of intermediate cells only in CONV and not GF mice. These findings provide evidence that manipulation and/or depletion of the enteric microbiota may have clinical significance in limiting chemotherapy-induced mucositis.}, number={5}, journal={Gut Microbes}, publisher={Informa UK Limited}, author={Rigby, Rachael J. and Carr, Jacquelyn and Orgel, Kelly and King, Stephanie L. and Lund, P. Kay and Dekaney, Christopher M.}, year={2016}, month={Jul}, pages={414–423} }
 @article{carr_king_dekaney_2015, title={1009 Depletion of Enteric Microbiota Protects from Chemotherapy-Induced Damage in the Murine Small Intestine}, volume={148}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(15)33862-2}, DOI={10.1016/S0016-5085(15)33862-2}, number={4}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Carr, Jacquelyn and King, Stephanie L and Dekaney, Christopher M.}, year={2015}, month={Apr}, pages={S-1132} }
 @article{king_carr_dekaney_2015, title={798 Mist1 Is Necessary for Paneth Cell Maturation}, volume={148}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(15)30527-8}, DOI={10.1016/S0016-5085(15)30527-8}, abstractNote={analysis, a Pre-Rockall score ‡4 (OR=4.647,95% CI : 1.432-15.086,p=0.005) and a Full-Rockall score ‡8 (OR=6.387,95% CI : 1.676-24.345,p=0.007) were independently associated with the high risk of death for patients with non-ulcer bleeding.A Glasgow-Blatchford score ‡ 6 (OR=14.996,95% CI : 6.141 -36.502, p<0.001) was a powerful predictor for the need for clinical intervention (blood transfusion, endoscopic hemostasis, surgery and angioembolization) in non-ulcer bleeding.CONCLUSIONS : Patients with non-ulcer bleeding have a similar clinical course as patients with ulcer bleeding.Therefore, we need to be more concerned about the use of scoring systems for the systematic management of non-ulcer bleeding.}, number={4}, journal={Gastroenterology}, publisher={Elsevier BV}, author={King, Stephanie L. and Carr, Jacquelyn and Dekaney, Christopher M.}, year={2015}, month={Apr}, pages={S-156-S-157} }
 @article{seiler_schenhals_von furstenberg_allena_smith_scaria_bresler_dekaney_henning_2015, title={Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage}, volume={361}, ISSN={0302-766X 1432-0878}, url={http://dx.doi.org/10.1007/s00441-015-2111-1}, DOI={10.1007/s00441-015-2111-1}, abstractNote={The goals of this study were to document the proliferative response of intestinal stem cells (ISCs) during regeneration after damage from doxorubicin (DXR), and to characterize the signals responsible for ISC activation. To this end, jejuni from DXR-treated mice were harvested for histology, assessment of ISC numbers and proliferation by flow cytometry, crypt culture, and RNA analyses. Histology showed that crypt depth and width were increased 4 days after DXR. At this time point, flow cytometry on tissue collected 1 h after EdU administration revealed increased numbers of CD24loUEA− ISCs and increased percentage of ISCs cycling. In culture, crypts harvested from DXR-treated mice were equally proliferative as those of control mice. Addition of subepithelial intestinal tissue (SET) collected 4 days after DXR elicited increased budding (1.4 ± 0.3 vs. 5.1 ± 1.0 buds per enteroid). Microarray analysis of SET collected 4 days after DXR revealed 1030 differentially expressed transcripts. Cross-comparison of Gene Ontology terms considered relevant to ISC activation pointed to 10 candidate genes. Of these, the epidermal growth factor (EGF) family member amphiregulin and the BMP antagonist chordin-like 2 were chosen for further study. In crypt culture, amphiregulin alone did not elicit significant budding, but amphiregulin in combination with BMP antagonism showed marked synergism (yielding 6.3 ± 0.5 buds per enteroid). These data suggest a critical role for underlying tissue in regulating ISC behavior after damage, and point to synergism between amphiregulin and chordin-like 2 as factors which may account for activation of ISCs in the regenerative phase.}, number={2}, journal={Cell and Tissue Research}, publisher={Springer Science and Business Media LLC}, author={Seiler, Kristen M and Schenhals, Erica L and von Furstenberg, Richard J and Allena, Bhavya K and Smith, Brian J and Scaria, Denny and Bresler, Michele N and Dekaney, Christopher M and Henning, Susan J}, year={2015}, month={Feb}, pages={427–438} }
 @article{gewain_king_dekaney_2013, title={Mo1824 Jejunal and Ileal Epithelium Demonstrate Differing Damage Responses to Doxorubicin Treatment}, volume={144}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(13)62492-0}, DOI={10.1016/S0016-5085(13)62492-0}, abstractNote={Introduction Intestinal fibrosis (IF) is a major complication of chronic inflammation of the intestinal tissue in inflammatory bowel disease (IBD).IF can cause narrowing of the intestinal lumen, which may lead to stricture formation.Currently, adequate models are lacking to study the mechanism of intestinal fibrosis in IBD.Our aim is to develop an ex vivo model for IF by using mouse precision-cut intestinal slice (PCIS).In addition, regional differences in the onset of fibrosis in the bowel will be studied.In PCIS all cell types are present in their original tissue-matrix environment.As fibrosis is a multicellular phenomenon, PCIS can be used as a model to study the early onset of intestinal fibrosis.Methods Mouse jejunum, ileum and colon were excised and prepared as segments embedded in agarose.PCIS (estimated 300-400 μm) were prepared and incubated up to 48 hr.ATP content of the PCIS was used to assess the general viability.To study the early onset of fibrosis, the gene expression of different fibrosis markers: Pro-Collagen 1 A1 (COL1A1), Heat Shock Protein 47 (HSP47), alpha-Smooth Muscle Actin (SMA) and Fibronectin (FN1) was determined in PCIS after 48 hr of incubation.Results Mouse PCIS from the different regions of the bowel were viable up to 48 hr in culture.After 48 hr of incubation of PCIS, HSP47 and FN1 gene expression was significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.9 and 3.6 fold, respectively) and in ileum (4.5 and 4.9 fold, respectively).Gene expression of Col1A1 (0.7 fold) was only down-regulated in PCIS from the colon.SMA gene expression was down-regulated in PCIS from all the regions of the intestine.PCIS from jejunum cultured in the presence of 5ng/mL Transforming-Growth Factor (TGF)-β1 for 48 hr, HSP47 (1.2 fold), FN1 (4.6 fold) and COL1A1 (2.0 fold) were significantly upregulated compared to control PCIS after 48 hr of incubation.In addition, in PCIS from Ileum incubated with 5ng/mL TGF-β1, the gene expression of HSP47 (1.9 fold) and FN1 (3.9 fold) was significantly increased.In PCIS from the colon cultured for 48 hr in the presence of 5ng/ml TGF-β1, the gene expression of fibrosis markers was not affected.Conclusion Mouse PCIS from different regions of the bowel can successfully be cultured for 48 hr.Fibrosis markers are induced by incubation up to 48 hr and by addition of TGF-β1 in jejunum and ileum PCIS, but not in colon PCIS.We are the first to show that regional differences in the onset of intestinal fibrosis can be measured using PCIS.}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Gewain, Kelly and King, Stephanie L. and Dekaney, Christopher M.}, year={2013}, month={May}, pages={S-672} }
 @article{king_mohiuddin_dekaney_2013, title={Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage}, volume={305}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00441.2012}, DOI={10.1152/ajpgi.00441.2012}, abstractNote={Paneth cell numbers increase following intestinal damage, but mechanisms driving this process are not understood. We hypothesized that the increase in Paneth cell numbers is due to recruitment of cells from a preexisting pool of secretory progenitors. Mice were given a single injection of doxorubicin (Dox), and intestinal tissue was collected 0-168 h after treatment. Paneth, goblet, and intermediate cells were counted and evaluated for cell morphology. Quantitative RT-PCR was used to measure expression of various genes associated with Paneth cell allocation and maturation. Paneth cells were birth dated using incorporation of thymidine analogs given before or after Dox. Staining revealed "intermediate" cells, which were rarely observed in control crypts but increased significantly in number 96 and 120 h after Dox treatment. Birth dating of intermediate cells 5 days after Dox treatment revealed that 24% of these cells took up thymidine analog given prior to Dox treatment and 36% took up thymidine analog given after Dox treatment. Quantitative RT-PCR demonstrated a significant increase in Spdef, Atoh1, Sox9, EphB3, Mist, Wnt5a, FGF-9, and FGF-18 mRNAs and a significant decrease in Indian hedgehog mRNA. Expansion of the Paneth cell compartment after Dox treatment is due to generation of new cells and recruitment of cells from an existing pool. These cells express Paneth and goblet biomarkers and are found only during repair. Expansion of these cells correlates temporally with reduced Indian hedgehog and increased FGF and Wnt mRNA. These findings are significant, as they provide a first step in understanding mechanisms of Paneth cell expansion during mucosal repair.}, number={2}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={King, Stephanie L. and Mohiuddin, Jahan J. and Dekaney, Christopher M.}, year={2013}, month={Jul}, pages={G151–G162} }
 @article{king_dekaney_2013, title={Small Intestinal Stem Cells}, volume={29}, journal={Current Opinion in Gastroenterology}, author={King, S.L. and Dekaney, C.M.}, year={2013}, pages={140–145} }
 @article{packey_gewain_sartor_dekaney_2013, title={Tu2027 The Chemotherapeutic Agent Doxorubicin Induces Contrasting Microbial Responses in the Jejunum and Distal Ileum That May Contribute to Differential Injury Patterns}, volume={144}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(13)63383-1}, DOI={10.1016/S0016-5085(13)63383-1}, abstractNote={Background.The protease inhibitor ritonavir is part of highly active anti-retroviral therapy (HAART) used successfully in the treatment of human immunodeficiency virus (HIV) infection.There is evidence that ritonavir alters intestinal permeability and induce damage to the small intestine.Because HIV infected patients taking HAART are at high risk of developing cardiovascular complications, there might be a need to use low dose of aspirin (ASA) to prevent inschemic event.Similarly, long term survival exposes HIV infects persons to detrimental interaction of ritonavir with NSAIDs, since both agents might cause intestinal injury.Aims.To test whether ritonavir worsens intestinal injury caused by NSAIDs and ASA.Methods.C57BL6 mice were treated for 25 days with ritonavir (50 mg/Kg/day per os) and than for others 5 days with the combination of ritonavir plus ASA (50 mg/Kg/day per os) or naproxen (100 mg/Kg/day per os).In a second set of experiments C57BL6 mice were treated for 25 days with ritonavir (50 mg/Kg/day per os) alone or in combination with the PGE 2 analog misoprostol (100 μg/Kg per os).Results.Ritonavir administration per se caused intestinal damage and its co-administration in combination with naproxen or ASA exacerbated the severity of intestinal damage and intestinal inflammation as assessed by measuring haematocrit, MPO activity, relative mRNA expression of iNOS, MCP-1 and VLA-1 (Figure ).All treatments caused reduced mucosal PGE 2 .Co-administration of misoprostol, a PGE 2 analogue, protected against intestinal damage induced by naproxen and ritonavir.Conclusions.The protease inhibitor ritonavir causes intestinal damage and its association with NSAIDs or ASA worsens damage caused by the COX-inhibitors.Misoprostol protects from damage caused by ritonavir.Further studies are need to clarify whether this observation has a clinical readout.}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Packey, Christopher D. and Gewain, Kelly and Sartor, Ryan B. and Dekaney, Christopher M.}, year={2013}, month={May}, pages={S-907-S-908} }
 @article{dehmer_garrison_speck_dekaney_henning_helmrath_2011, title={Characterization Of Intestinal Stem Cells During Development: The Role Of Lgr5}, volume={165}, ISSN={0022-4804}, url={http://dx.doi.org/10.1016/j.jss.2010.11.142}, DOI={10.1016/j.jss.2010.11.142}, number={2}, journal={Journal of Surgical Research}, publisher={Elsevier BV}, author={Dehmer, J.J. and Garrison, A.P. and Speck, K.E. and Dekaney, C.M. and Henning, S.J. and Helmrath, M.A.}, year={2011}, month={Feb}, pages={339} }
 @article{dehmer_garrison_speck_dekaney_van landeghem_sun_henning_helmrath_2011, title={Expansion of Intestinal Epithelial Stem Cells during Murine Development}, volume={6}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0027070}, DOI={10.1371/journal.pone.0027070}, abstractNote={Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.}, number={11}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Dehmer, Jeffrey J. and Garrison, Aaron P. and Speck, Karen E. and Dekaney, Christopher M. and Van Landeghem, Laurianne and Sun, Xiaofei and Henning, Susan J. and Helmrath, Michael A.}, editor={Rota, MarcelloEditor}, year={2011}, month={Nov}, pages={e27070} }
 @article{rigby_lund_dekaney_2010, title={750 Germ Free Mice Show Apoptosis but Dramatically Reduced Overt Mucosal Damage After Doxorubicin}, volume={138}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(10)60471-4}, DOI={10.1016/S0016-5085(10)60471-4}, abstractNote={contributing 11.053 person-years of follow-up.During the study period, a total of 27 CRC cases occurred among FDRs at a median age of 62 years (interquartile range: 57-78) compared to 5 expected CRC cases (p<0.001).The relative risk of CRC in FDRs compared to the general population was 5.4 (95%-CI: 3.7-7.8).No significant difference was seen between the relative risk for male and female FDRs regarding the CRC incidence.In 4 FDRs from 4 different pedigrees multiple histologically confirmed hyperplastic polyps were reported, satisfying the criteria for HPS.Based on the estimated HPS prevalence of 1:3000 in the general population the projected relative risk of HPS in FDRs would be 39 (95%-CI: 13-121).In most FDRs endoscopy was not performed (281/347: 81%) suggesting that the true prevalence of CRC and HPS in FDRs may be underestimated.Conclusions: Our results showed that FDRs of HPS patients have an increased relative risk for both CRC and HPS.Hence, as long as no genetic substrate has been identified, screening colonoscopies for FDRs seem justified but needs to be prospectively evaluated.}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Rigby, Rachael J. and Lund, Pauline K. and Dekaney, Christopher M.}, year={2010}, month={May}, pages={S-103} }
 @article{dekaney_2010, title={T1727 Paneth Cells Expand From a Pre-Existing Pool During Repair After Doxorubicin-Induced Damage}, volume={138}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(10)62605-4}, DOI={10.1016/S0016-5085(10)62605-4}, abstractNote={types are only partially understood.Aim: in the current study, we evaluated the role of β2SP (β2 spectrin) in the differentiation and asymmetric division of stem/progenitor cells in normal intestine epithelium.Results: Our preliminary data showed that the number of secreting-goblet cells and peptide hormone secreting enteroendocrine cells was reduced in β2SP+/mice compared to the wt mice.The number of cells undergo asymmetric division was also 3 folds lower in β2SP+/mice than in wt mice.In In Vitro studies, polarization of Par3 and PKCζ was impaired in β2SP-/-MEF cells.The coimmunoprecipitation assay demonstrated that β2SP directly bound to Par3 and actin in normal intestine epithelial cells, indicating that β2SP was involved in the spindle orientation and positioning during the asymmetric division of intestine epithelial cells.Conclusion: β2SP interacts with Par3 and regulates spindle orientation and polarization in the process of cell division, thereby contributing to the differentiation and asymmetric division of intestinal stem/progenitor cells.}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Dekaney, Christopher M.}, year={2010}, month={May}, pages={S-566} }
 @article{dekaney_gulati_helmrath_henning_2009, title={488 Regeneration of Intestinal Stem Cells Following Doxorubicin Treatment of Mice}, volume={136}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(09)60354-1}, DOI={10.1016/S0016-5085(09)60354-1}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Dekaney, Christopher M. and Gulati, Ajay S. and Helmrath, Michael A. and Henning, Susan J.}, year={2009}, month={May}, pages={A-79} }
 @article{garrison_dekaney_von allmen_lund_henning_helmrath_2009, title={Early but not late administration of glucagon-like peptide-2 following ileo-cecal resection augments putative intestinal stem cell expansion}, volume={296}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.90588.2008}, DOI={10.1152/ajpgi.90588.2008}, abstractNote={Expansion of intestinal progenitors and putative stem cells (pISC) occurs early and transiently following ileo-cecal resection (ICR). The mechanism controlling this process is not defined. We hypothesized that glucagon-like peptide-2 (GLP-2) would augment jejunal pISC expansion only when administered to mice immediately after ICR. Since recent reports demonstrated increases in intestinal insulin-like growth factor (IGF)-I following GLP-2 administration, we further hypothesized that increased intestinal IGF-I expression would correlate with pISC expansion following ICR. To assess this, GLP-2 or vehicle was administered to mice either immediately after resection (early) or before tissue harvest 6 wk following ICR (late). Histological analysis quantified proliferation and intestinal morphometrics. Serum levels of GLP-2 were measured by ELISA and jejunal IGF-I mRNA by qRT-PCR. Expansion of jejunal pISC was assessed by fluorescent-activated cell sorting of side population cells, immunohistochemistry for phosphorylated beta-catenin at serine 552 (a pISC marker), percent of crypt fission, and total numbers of crypts per jejunal circumference. We found that early but not late GLP-2 treatment after ICR significantly augmented pISC expansion. Increases in jejunal IGF-I mRNA correlated temporally with early pISC expansion and effects of GLP-2. Early GLP-2 increased crypt fission and accelerated adaptive increases in crypt number and intestinal caliber. GLP-2 increased proliferation and intestinal morphometrics in all groups. This study shows that, in mice, GLP-2 promotes jejunal pISC expansion only in the period immediately following ICR. This is associated with increased IGF-I and accelerated adaptive increases in mucosal mass. These data provide clinical rationale relevant to the optimal timing of GLP-2 in patients with intestinal failure.}, number={3}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Garrison, Aaron P. and Dekaney, Christopher M. and von Allmen, Douglas C. and Lund, P. Kay and Henning, Susan J. and Helmrath, Michael A.}, year={2009}, month={Mar}, pages={G643–G650} }
 @article{dekaney_gulati_garrison_helmrath_henning_2009, title={Regeneration of intestinal stem/progenitor cells following doxorubicin treatment of mice}, volume={297}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.90446.2008}, DOI={10.1152/ajpgi.90446.2008}, abstractNote={The intestinal epithelium is in a constant state of renewal. The rapid turnover of cells is fed by a hierarchy of transit amplifying and stem/progenitor cells destined to give rise to the four differentiated epithelial lineages of the small intestine. Doxorubicin (Dox) is a commonly used chemotherapeutic agent that preferentially induces apoptosis in the intestinal stem cell zone (SCZ). We hypothesized that Dox treatment would initially decrease "+4" intestinal stem cell numbers with a subsequent expansion during mucosal repair. Temporal assessment following Dox treatment demonstrated rapid induction of apoptosis in the SCZ leading to a decrease in the number of intestinal stem/progenitor cells as determined by flow cytometry for CD45(-) SP cells, and immunohistochemistry of cells positive for putative +4 stem cell markers beta-cat(Ser552) and DCAMKL1. Between 96 and 168 h postinjection, overall proliferation in the crypts increased concomitant with increases in both absolute and relative numbers of goblet, Paneth, and enteroendocrine cells. This regeneration phase was also associated with increases of CD45(-) SP cells, beta-cat(Ser552)-positive cells, crypt fission, and crypt number. We used Lgr5-lacZ mice to assess behavior of Lgr5-positive stem cells following Dox and found no change in this cell population. Lgr5 mRNA level was also measured and showed no change immediately after Dox but decreased during the regeneration phase. Together these data suggest that, following Dox-induced injury, expansion of intestinal stem cells occurs during mucosal repair. On the basis of available markers this expansion appears to be predominantly the +4 stem cell population rather than those of the crypt base.}, number={3}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Dekaney, Christopher M. and Gulati, Ajay S. and Garrison, Aaron P. and Helmrath, Michael A. and Henning, Susan J.}, year={2009}, month={Sep}, pages={G461–G470} }
 @article{garrison_dekaney_von allmen_henning_helmrath_2008, title={163. Timing of Glucagon-Like Peptide-2 (GLP-2) Required for Augmented Expansion of Intestinal Stem Cells Following Ileo-Cecal Resection (ICR) in Mice}, volume={144}, ISSN={0022-4804}, url={http://dx.doi.org/10.1016/j.jss.2007.12.186}, DOI={10.1016/j.jss.2007.12.186}, abstractNote={Expansion of intestinal stem cells leading to new crypt formation is an important long-term intestinal adaptive mechanism following ileo-cecal resection (ICR) in mice. Clinical experience with administration of growth factors does not appear to result in many of the acute responses seen in animal models. We hypothesize that timing of growth factor administration following intestinal loss may affect the expansion of intestinal stem cells. In this study, we sought to compare both early and late treatment of GLP-2 on intestinal morphology, proliferation, expansion of intestinal stem cells, and differentiated lineages following ICR. Methods: All mice underwent ICR and were randomized to either early (1 week) or late (6 week) groups. Mice within each group received GLP-2 or vehicle IP every 12 hours for 7 days (n = 6-10/group). Expansion of intestinal stem cells was measured by increases in crypt fission and assayed by flow cytometry. Early stem cell expansion was confirmed in additional groups by BrdU label retention. Immunohistochemistry and H&E of jejunum were used to evaluate changes in intestinal morphometrics and allocation of intestinal lineages. Means were compared using ANOVA and Fisher’s procedure. Results: Early administration of GLP-2 significantly increased the percent of putative stem cells (SP) and rates of crypt fission resulting in marked increases in the number of crypts per bowel circumference compared to vehicle and late GLP-2 treated mice. Label retention studies confirmed these findings. Although similar increases in proliferation, villus height, and crypt depth occurred in both early and late groups, significant changes in the allocation of Goblet cells (increased) and Paneth cells (decreased) only occurred following early administration of GLP-2, highlighting specific differences in timing. Please see Table 1 where “a” denotes differences between GLP-2 vs. vehicle treated at the same timepoints, and “b” denotes differences between early vs. late treatment groups (p < 0.05). Conclusion: Although GLP-2 has many intestinal trophic effects, expansion of intestinal stem cells appears to occur only during a brief time frame following ICR. Insight into the timing of growth factor therapy may be clinically relevant. Further studies identifying the mechanisms expanding stem cells may provide clues to the management of chronic intestinal failure. TABLE 1 Treatment Group %SP % Crypt Fission #Crypts/circum-ference Proliferative Index Villus Height (μm) Crypt Depth (μm) % Goblet cells % Paneth cells 1 week ICR + vehicle 1.46 ± 1.0 10.5 ± 3% 146 ± 21 31.5 ± 2 327 ± 27 110 ± 10 7.7 ± 1% 19.4 ± 3% Early 1 week ICR + GLP-2 3.14 ± 0.8a 16.8 ± 3%a 248 ± 45a 41.0 ± 5a 465 ± 61a 126 ± 18 10.3 ± 2%a 14.6 ± 2%a 6 weeks ICR + vehicle .07 ± .01 6.4 ± 2% 313 ± 85 27.7 ± NA 459 ± 63 145 ± 25 6.2 ± 1% 13.8 ± 1% Late 6 weeks ICR + GLP-2 .14 ± .04b 6.3 ± 2%b 318 ± 50b 43.4 ± 4a 544 ± 62a,b 166 ± 16b 6.2 ± 1%b 13.6 ± 2%b Open table in a new tab}, number={2}, journal={Journal of Surgical Research}, publisher={Elsevier BV}, author={Garrison, Aaron P. and Dekaney, Christopher M. and von Allmen, Douglas C. and Henning, Susan J. and Helmrath, Michael A.}, year={2008}, month={Feb}, pages={249–250} }
 @article{dekaney_von allmen_garrison_rigby_lund_henning_helmrath_2008, title={29. Bacterial Dependent up Regulation of Ileal Bile Acid Binding Protein and Bile Acid Transporters Is FXR-Mediated Following Ileo-Cecal Resection (ICR)}, volume={144}, ISSN={0022-4804}, url={http://dx.doi.org/10.1016/j.jss.2007.12.034}, DOI={10.1016/j.jss.2007.12.034}, abstractNote={Enteric microbiota may exacerbate chronic inflammation in patients with Crohn’s disease. In these patients, recurrent disease often results in surgical resection of the terminal ileum, disrupting the normal enterohepatic circulation of bile acids. The ability of the colon to up regulate the molecular mechanisms required for bile acid reclamation following ICR helps prevent colonic mucosa from chronic injury due to increased luminal bile acid content. In this study, we hypothesized that enteric microbiota are required for luminal bile salts to up regulate FXR-mediated bile acid transport in the colon. Methods: To test our hypothesis, conventional (WT), conventional FXR knockout (FXR −/−) and germ free (GF) mice maintained throughout the study in a gnotobiotic facility were randomized to undergo either ICR or sham operation (n = 3-6/group). On postoperative day 7 the ascending colon was harvested for luminal contents, histology, immunohistochemistry and isolation of total RNA and protein. Real time RT-PCR was performed to evaluate changes in bile acid homeostatic gene expression of IBABP (ileal bile acid binding protein), Asbt (apical bile acid transporter), Ostβ (organic solute transporter) and FGF 15 (regulates liver bile homeostasis) in the colon. Means were compared using ANOVA and Fisher’s pairwise comparison. Results: The lack of microbiota in GF mice resulted in significant increases in colonic crypt depth following ICR compared to sham operated animals, whereas no differences were noted in WT mice. Increases in the expression of genes regulating bile acid transporters were observed for IBABP (12 fold), Asbt (5.6 fold), Ostβ (1.6 fold) and FGF 15 (5.9 fold) only in WT mice following ICR. Increased expression of IBABP was confirmed by immunohistochemistry. FXR (−/−) mice demonstrated similar histological changes as occurred in GF mice. Consistent with this observation was the lack of up regulation of IBABP and FGF 15 following ICR, whereas similar increases in Asbt (3 fold) and Ostβ (2 fold) occurred as in WT mice. Please see Table 1 where “a” denotes differences between sham and ICR of a given mouse type, and “b” denotes differences between either FXR(−/−) or GF mice and WT mice for a given surgery. Conclusion: Bacterial dependent up regulation of IBABP is FXR mediated in the colon following ICR. Mice lacking microbiota (GF) or FXR are unable to increase the expression of IBABP or FGF 15, resulting in colonic crypt depth in response to increased luminal bile acids. Increases in Asbt and Ostβ require luminal bacteria, but do not appear to be FXR mediated. Further insight into colonic adaptation to increased bile acids may prove beneficial to managing Crohn’s patients following surgical loss of the ileum. Tabled 1 Mouse Type Surgery Colonic Crypt Depth Relative Gene Expression IBABP FGF 15 Asbt Ost β WT Sham 94.3 ± 5.8 4.8 ± 1.6 0.4 ± 0.3 0.2 ± 0.05 1.2 ± 0.2 WT ICR 104.9 ± 2.6 57.8 ± 15.1a 2.4 ± 0.9 0.8 ± 0.4 1.9 ± 0.5 FXR −/− Sham 93.7 ± 1.9 0.6 ± 0.1 0.002 ± 0.001 0.7 ± 0.1 1.6 ± 0.1 FXR −/− ICR 116.3 ± 2.6a 0.3 ± 0.03b 0.05 ± 0.03 2.2 ± 0.1a,b 2.3 ± 0.1 GF Sham 92.5 ± 3.1 9.1 ± 8.2 13.5 ± 7.7a 0.8 ± 0.5 0.3 ± 0.04 GF ICR 145.5 ± 12.3a,b 1.6 ± 0.2b 3.5 ± 1.7 0.1 ± 0.02 0.3 ± 0.07b Open table in a new tab}, number={2}, journal={Journal of Surgical Research}, publisher={Elsevier BV}, author={Dekaney, Chrisopher M. and von Allmen, Doug C. and Garrison, Aaron P. and Rigby, Rachael J. and Lund, P. Kay and Henning, Susan J. and Helmrath, Michael A.}, year={2008}, month={Feb}, pages={188–189} }
 @article{dekaney_von allmen_garrison_rigby_lund_henning_helmrath_2008, title={Bacterial-dependent up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection}, volume={144}, ISSN={0039-6060}, url={http://dx.doi.org/10.1016/j.surg.2008.03.035}, DOI={10.1016/j.surg.2008.03.035}, abstractNote={Background Bile acid (BA) reclamation following ileo-cecal resection (ICR) may prevent colonic mucosa from chronic injury. In this study, we hypothesized that in a murine model of ICR the remnant colon would upregulate the cellular machinery necessary for BA reclamation and would do so in an FXR- and bacteria-dependent manner. Methods Conventional (WT), conventional FXR knockout (FXR null) and germ-free (GF) mice were randomized to undergo either ICR or sham operation. The ascending colon was harvested for histology and immunohistochemistry and changes in bile acid homeostatic gene expression determined by real-time polymerase chain reaction (RT-PCR) 7 days following surgery. Results Following ICR WT mice showed significant increases in the expression of genes regulating bile acid transport including IBABP, Asbt, Ostβ and FGF 15. Increased expression of IBABP and Asbt was confirmed by immunohistochemistry. Induction of bile acid transport genes was absent or attenuated in FXR null and GF mice. Conclusion Bacterial dependent up regulation of IBABP is FXR mediated in the colon following ICR. Mice lacking microbiota (GF) or FXR are unable to increase the expression of IBABP or FGF 15. Bile acid (BA) reclamation following ileo-cecal resection (ICR) may prevent colonic mucosa from chronic injury. In this study, we hypothesized that in a murine model of ICR the remnant colon would upregulate the cellular machinery necessary for BA reclamation and would do so in an FXR- and bacteria-dependent manner. Conventional (WT), conventional FXR knockout (FXR null) and germ-free (GF) mice were randomized to undergo either ICR or sham operation. The ascending colon was harvested for histology and immunohistochemistry and changes in bile acid homeostatic gene expression determined by real-time polymerase chain reaction (RT-PCR) 7 days following surgery. Following ICR WT mice showed significant increases in the expression of genes regulating bile acid transport including IBABP, Asbt, Ostβ and FGF 15. Increased expression of IBABP and Asbt was confirmed by immunohistochemistry. Induction of bile acid transport genes was absent or attenuated in FXR null and GF mice. Bacterial dependent up regulation of IBABP is FXR mediated in the colon following ICR. Mice lacking microbiota (GF) or FXR are unable to increase the expression of IBABP or FGF 15.}, number={2}, journal={Surgery}, publisher={Elsevier BV}, author={Dekaney, Christopher M. and von Allmen, Douglas C. and Garrison, Aaron P. and Rigby, Rachael J. and Lund, P. Kay and Henning, Susan J. and Helmrath, Michael A.}, year={2008}, month={Aug}, pages={174–181} }
 @article{garrison_dekaney_von allmen_henning_helmrath_2008, title={Developmental insight into intestinal adaptation: characterization of intestinal stem cell (ISC) expansion and glucagon-like peptide-2 (GLP-2) actions during murine intestinal development}, volume={207}, ISSN={1072-7515}, url={http://dx.doi.org/10.1016/j.jamcollsurg.2008.06.120}, DOI={10.1016/j.jamcollsurg.2008.06.120}, abstractNote={Garrison, Aaron P. MD; Dekaney, Christopher M. PhD; von Allmen, Douglas C. BS; Henning, Susan J. PhD; Helmrath, Michael A. MD Author Information}, number={3}, journal={Journal of the American College of Surgeons}, publisher={Elsevier BV}, author={Garrison, Aaron P. and Dekaney, Christopher M. and von Allmen, Douglas C. and Henning, Susan J. and Helmrath, Michael A.}, year={2008}, month={Sep}, pages={S55–S56} }
 @article{dekaney_wu_yin_jaeger_2008, title={Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells}, volume={19}, ISSN={0955-2863}, url={http://dx.doi.org/10.1016/j.jnutbio.2007.09.002}, DOI={10.1016/j.jnutbio.2007.09.002}, abstractNote={Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12–72 h in the presence of 0, 0.01 and 1 μM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 μM RA, addition of 10 μM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.}, number={10}, journal={The Journal of Nutritional Biochemistry}, publisher={Elsevier BV}, author={Dekaney, Christopher M. and Wu, Guoyao and Yin, Yu-Long and Jaeger, Laurie A.}, year={2008}, month={Oct}, pages={674–681} }
 @article{garrison_dekaney_von allmen_lund_henning_helmrath_2008, title={T1818 Early Administration of Glucagon-Like Peptide-2 (GLP-2) Following Ileocecal Resection Augments Insulin-Like Growth Factor-I (IGF-I) Signaling During Intestinal Stem Cell Expansion}, volume={134}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(08)62659-1}, DOI={10.1016/S0016-5085(08)62659-1}, abstractNote={antibody recognizing the C-terminus, a lower molecular weight species of 45-kDa was observed in addition to the full-length 66-kDa SHP-1 protein.Treatment with MG132 led to an increase in expression of full-length SHP-1 protein and concomitantly led to a decrease in expression levels of the lower molecular species of 45-kDa.7-Western blot analyses revealed that the 45-kDa protein is a peptide fragment corresponding to the C-terminal portion of SHP-1 generated from proteasome activity.8-By using Pcleavage, a support vector machine based method for prediction of proteasome cleavage sites (www.Imtech.res.in/cgibin/pcleavage/), we found that the Y208 might be a proteasome cleavage site in SHP-1 coding sequence.Mutational analysis of Y208 residue on SHP-1 abolished expression of the carboxy-truncated 45-kDa SHP-1 protein.Conclusion.Our results indicate that the abundance of SHP-1 protein is regulated by post-translational (proteolysis) pathways.Cdk2associated complexes, by targeting SHP-1 for degradation, might counteract the ability of SHP-1 to block cell cycle progression of IECs.}, number={4}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Garrison, Aaron P. and Dekaney, Christopher M. and von Allmen, Doug C. and Lund, P. Kay and Henning, Susan J. and Helmrath, Michael A.}, year={2008}, month={Apr}, pages={A-569-A-570} }
 @article{dekaney_fong_rigby_lund_henning_helmrath_2007, title={Expansion of intestinal stem cells associated with long-term adaptation following ileocecal resection in mice}, volume={293}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00218.2007}, DOI={10.1152/ajpgi.00218.2007}, abstractNote={Sustained increases in mucosal surface area occur in remaining bowel following massive intestinal loss. The mechanisms responsible for expanding and perpetuating this response are not presently understood. We hypothesized that an increase in the number of intestinal stem cells (ISC) occurs following intestinal resection and is an important component of the adaptive response in mice. This was assessed in the jejunum of mice 2-3 days, 4-5 days, 6-7 days, 2 wk, 6 wk, and 16 wk following ileocecal resection (ICR) or sham operation. Changes in ISC following ICR compared with sham resulted in increased crypt fission and were assayed by 1) putative ISC population (SP) by flow cytometry, 2) Musashi-1 immunohistochemistry, and 3) bromodeoxyuridine (BrdU) label retention. Observed early increases in crypt depth and villus height were not sustained 16 wk following operation. In contrast, long-term increases in intestinal caliber and overall number of crypts per circumference appear to account for the enhanced mucosal surface area following ICR. Flow cytometry demonstrated that significant increases in SP cells occur within 2-3 days following resection. By 7 days, ICR resulted in marked increases in crypt fission and Musashi-1 immunohistochemistry staining. Separate label-retention studies confirmed a 20-fold increase in BrdU incorporation 6 wk following ICR, confirming an overall increase in the number of ISC. These studies support that expansion of ISC occurs following ICR, leading to an overall increase number of crypts through a process of fission and intestinal dilation. Understanding the mechanism expanding ISCs may provide important insight into management of intestinal failure.}, number={5}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Dekaney, Christopher M. and Fong, Jerry J. and Rigby, Rachael J. and Lund, P. Kay and Henning, Susan J. and Helmrath, Michael A.}, year={2007}, month={Nov}, pages={G1013–G1022} }
 @article{helmrath_fong_dekaney_henning_2007, title={Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice}, volume={292}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00188.2006}, DOI={10.1152/ajpgi.00188.2006}, abstractNote={Following massive small bowel resection (SBR) in mice, there are sustained increases in crypt depth and villus height, resulting in enhanced mucosal surface area. The early mechanisms responsible for resetting and sustaining this increase are presently not understood. We hypothesized that expansion of secretory lineages is an early and sustained component of the adaptive response. This was assessed in the ileum by quantitative morphometry at 12 h, 36 h, 7 days, and 28 days and by quantitative RT-PCR of marker mRNAs for proliferation and differentiated goblet, Paneth cell, and enterocyte genes at 12 h after 50% SBR or sham operation. As predicted, SBR elicited increases of both crypt and villus epithelial cells, which were sustained though the 28 days of the experiment. Significant increases in the overall number and percentage of both Paneth and goblet cells within intestinal epithelium occurred by 12 h and were sustained up to 28 days after SBR. The increases of goblet cells after SBR were initially observed within villi at 12 h, with marked increases occurring in crypts at 36 h and 7 days. Consistent with this finding, qRT-PCR demonstrated significant increases in the expression of mRNAs associated with proliferation (c-myc) and differentiated goblet cells (Tff3, Muc2) and Paneth cells (lysozyme), whereas mRNA associated with differentiated enterocytes (sucrase-isomaltase) remained unchanged. From these data, we speculate that early expansion of intestinal secretory lineages within the epithelium of the ileum occurs following SBR, possibly serving to amplify the signal responsible for initiating and sustaining intestinal adaptation.}, number={1}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Helmrath, Michael A. and Fong, Jerry J. and Dekaney, Christopher M. and Henning, Susan J.}, year={2007}, month={Jan}, pages={G215–G222} }
 @article{dekaney_fong_rodriguez_henning_helmrath_2006, title={Massive small bowel resection results in sustained increases in intestinal stem cell number}, volume={130}, ISSN={0022-4804}, url={http://dx.doi.org/10.1016/j.jss.2005.11.262}, DOI={10.1016/j.jss.2005.11.262}, abstractNote={Introduction: Prolonged increases in proliferation of the intestinal epithelium have been demonstrated following massive small bowel resection (SBR) resulting in sustained increases in villus height and crypt depth. The mechanism of this action may be explained by either 1) increases in the number of intestinal stem cells or 2) increased proliferation by the same number of stem cells. Currently there are no techniques for isolating intestinal stem cells. Our lab has recently published flow cytometric methodology for isolating an intestinal side population (SP) which is enriched for putative intestinal stem cells. In addition, expression of the RNA binding protein Musashi-1 by immunohistochemistry within the stem cell region as well as enhancement of Musashi-1 in our SP population support its possible role as an intestinal stem cell marker. In this study we sought to investigate the effects of SBR on these markers of intestinal stem cells. We hypothesize that the adaptive response following SBR is the result of increased number of intestinal stem cells. Methods: The terminal ileum from male C57/Bl6 mice (n=3 for each time point) from sham operated or 50% SBR animals was collected at 2 days, 5 days, and 14 days post-surgery. Flow cytometry was used to evaluate changes in the side population and immunohistochemical analysis was used to investigate Musashi-1 protein expression. Results: By 5 days post surgery, flow cytometric analysis revealed a greater than 3-fold increase in the percentage of SP cells in the terminal ileum from SBR animals compared to sham operated animals suggesting an increase in the number of intestinal stem cells. As expected the percentage of SP cells decreased as total epithelium expands due to adaptation by 14 days. Immunohistochemistry of Musashi-1 demonstrates sustained expansion of the intestinal stem cell zone from SBR animals at all time points beyond 5 days. Conclusions: These data suggest that intestinal stem cell numbers are increased in ileal epithelium by 5 days in response to SBR. Expansion of the stem cell zone as demonstrated by immunohistochemistry of Musashi-1 supports the increased number of intestinal stem cells as the mechanism responsible for sustained proliferation following SBR. Tabled 1Percent SP cells 2 Days 5 Days 14 Days Sham 0.07% ± 0.01% 0.16% ± 0.01% 0.60% ± 0.12% SBR 0.06% ± 0.01% 0.90% ± 0.12% ⁎ p < 0.05 compared to 5 Day Sham 0.38% ± 0.15% value = average percent ± standard error p < 0.05 compared to 5 Day Sham Open table in a new tab value = average percent ± standard error}, number={2}, journal={Journal of Surgical Research}, publisher={Elsevier BV}, author={Dekaney, C.M. and Fong, J. and Rodriguez, J.M. and Henning, S.J. and Helmrath, M.A.}, year={2006}, month={Feb}, pages={253} }
 @article{dekaney_rodriguez_graul_henning_2006, title={Reply}, volume={130}, ISSN={0016-5085}, url={http://dx.doi.org/10.1053/J.GASTRO.2006.01.076}, DOI={10.1053/J.GASTRO.2006.01.076}, abstractNote={We thank Alison et al for their comments, which have given us the opportunity to discuss some important issues related to our paper. First, we would like to emphasize that in all tissues the study of stem cells has been greatly facilitated by the development of methods for sorting viable cells. Thus, the fact that bone marrow hematopoietic stem cells are by far the best characterized stem cell population reflects the ease of obtaining single cell suspensions from bone marrow. For the small intestinal epithelium our paper is, to our knowledge, the first published description of methods for generating viable single cell suspensions suitable for cell sorting and the first reported isolation of a viable subpopulation enriched for putative intestinal epithelial stem cells. At first glance the paper by Asakura and Rudnicki1Asakura A. Rudnicki M.A. Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation.Exp Hematol. 2002; 30: 1339-1345Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar may appear to be a precedent; however, the key issue is that the purpose of that study was to isolate CD45-positive SP cells from various tissues and to test their hematopoietic potential. Although these authors reported in Table 3 that 77% of the intestinal SP was CD45-negative, they made no further investigation of, nor comment on, these cells. Based on our experience we believe that the digestion conditions used would have released epithelial cells as sheets rather than single cells. Thus, these CD45-negative intestinal SP cells were most probably derived from the lamina propria rather than the epithelium and are not equivalent to those reported in our paper. Alison et al had 3 concerns regarding our conclusion that our CD45-negative SP preparations are enriched for epithelial stem cells. Their first concern related to markers. The critical issue here is that, at the time this manuscript was submitted, Musashi-1 was the best candidate marker for intestinal epithelial stem cells. Based on the fact that Musashi-1 mRNA was enriched in the CD45-negative SP fraction (Figure 8) and not in any other fraction (data not shown), we still believe it is reasonable to conclude that the cells expressing Musashi-1 are captured in the SP. In view of the presence of lineage markers, we recognized that the preparations are “not by any means pure” and that the EDTA/SP almost certainly includes “significant numbers of transit amplifying cells” (Discussion p. 1578). This is tantamount to saying that the preparation is enriched in crypt-base cells as Alison et al suggest. Based on the known expression of several ABC transporters (including ABCG2 which is also known as Bcrp1) on the brush border of villus epithelial cells, we, like Alison et al were initially concerned that these cells would efflux Hoechst and thus fractionate into the SP. On closer examination of the literature, however, we discovered that expression of ABCG2 is not sufficient to cause cells to sort into the SP. Thus for example, Zhou et al2Zhou S. Schuetz J.D. Bunting K.D. Colapietro A.M. Sampath J. Morris J.J. Lagutina I. Grosveld G.C. Osawa M. Nakauchi H. Sorrentino B.P. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.Nat Med. 2001; 7: 1028-1034Crossref PubMed Scopus (1992) Google Scholar found that murine ES cells displayed equivalent expression of ABCG2 in SP and non-SP fraction and concluded that “the SP phenotype requires additional cellular characteristics aside from Bcrp1 expression.” In our study, if ABCG2 on villus epithelial cells caused them to sort into the SP, the proportion of cells found in this fraction would have been very much higher than the 1%–2% we reported. The final point is that we purposefully called these cells “putative intestinal stem cells” (PISC), in the title and throughout the paper, in acknowledgment of the fact that, as yet, we have no functional evidence that they are indeed stem cells. It is our belief that “definitive proof” related to this or any other preparation of PISC, will require the efforts of many laboratories and that our report should be viewed merely as a “valuable first step” (Discussion p. 1578). For the small intestine, lack of methods for even generating viable single cell suspensions has been a major impediment to the study of epithelial stem cells. In publishing our paper we had hoped to stimulate the field by enabling other investigators to explore alternate sorting strategies and to develop much-needed functional assays. We ourselves are further characterizing the CD45-negative SP by microarray analysis, at the same time as exploring both in vivo and in vitro conditions which will allow assessment of “stemness.” Isolation of Gut SP Cells Does Not Automatically Enrich for Stem CellsGastroenterologyVol. 130Issue 3PreviewWe read with great interest the article by Dekaney et al1 describing the survivability (for 2 weeks in culture) of an SP fraction isolated from normal and neonatal mouse jejunum. This achievement confirms earlier but uncited data of Asakura and Rudnicki2 who established that there was a substantial verapamil-sensitive Hoechst 33342 side population (∼8%) in cells dissociated from adult mouse small intestine; they further tested their hematopoietic potential in culture and reported that three quarters of the SP was CD45-negative. Full-Text PDF}, number={3}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Dekaney, Christopher M. and Rodriguez, Jose M. and Graul, M. Colleen and Henning, Susan J.}, year={2006}, month={Mar}, pages={1013–1014} }
 @article{dekaney_rodriguez_graul_henning_2005, title={Isolation and Characterization of a Putative Intestinal Stem Cell Fraction From Mouse Jejunum}, volume={129}, ISSN={0016-5085}, url={http://dx.doi.org/10.1053/j.gastro.2005.08.011}, DOI={10.1053/j.gastro.2005.08.011}, abstractNote={BACKGROUND & AIMS
Although there have been many recent advances regarding the biology of intestinal stem cells, the field has been hampered significantly by the lack of a method to isolate these cells. Therefore, the aim of this study was to explore the hypothesis that viable intestinal stem cells can be isolated as a side population (SP) by fluorescence-activated cell sorting after staining with the DNA-binding dye Hoechst 33342.


METHODS
Preparations of individual cells from either whole mucosa or epithelium of mouse jejunum were stained with Hoechst 33342 and propidium iodide and then sorted using fluorescence-activated cell sorting. Cells were characterized using fluorochrome-labeled antibodies to surface markers, intracellular markers, and annexin V to detect early apoptosis. Total RNA was isolated from sorted fractions and used for quantitative real-time reverse-transcription polymerase chain reaction to evaluate the expression of cell lineage markers and the intestinal stem-cell marker, Musashi-1.


RESULTS
Adult and neonatal jejunum contain a viable population of cells that shows the SP phenotype and is sensitive to verapamil. This population of cells (from both mucosal and epithelial preparations) includes a CD45-negative fraction corresponding to nonhematopoietic cells, which shows minimal expression of surface markers typically found on stem cells from other tissues and of intracellular markers found in mesenchymal cells. Additionally, these cells were enriched for Musashi-1 and beta1-integrin, were cytokeratin positive, and survived in culture for up to 14 days.


CONCLUSIONS
The CD45-negative SP fraction, although not pure, represents the successful isolation of a viable population significantly enriched in small intestinal epithelial stem cells.}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Dekaney, Christopher M. and Rodriguez, Jose M. and Graul, M. Colleen and Henning, Susan J.}, year={2005}, month={Nov}, pages={1567–1580} }
 @article{helmrath_fong_dekaney_henning_2004, title={Early activation of Wnt β-catenin system following a massive small bowel resection in mice}, volume={121}, ISSN={0022-4804}, url={http://dx.doi.org/10.1016/j.jss.2004.07.140}, DOI={10.1016/j.jss.2004.07.140}, abstractNote={Introduction. Recent evidence supports a role for the Wnt β-catenin system in the regulation of intestinal proliferation, migration, and differentiation towards secretory lineages in mature epithelium. The observation that early increased allocation of secretory lineages (Paneth, goblet, and enteroendocrine cells) within intestinal epithelium occurs following a massive small bowel resection (SBR) suggest activation of the Wnt β-catenin system may initiate the adaptive response following resection. The purpose of this study was to determine early changes in gene expression of the soluble intestinal Wnts and their downstream targets responsible for proliferation (c-myc), migration (EphrinB1 and B2), and Paneth cell differentiation (EphB3). Methods. Male mice (C57/BL6J, 22–25 g) were randomized to either a sham operation (transection and reanastomosis) or 50% SBR (mid-jejunum to proximal ileum). All animals (n = 5–8 per group) were maintained on liquid diet 12 h following surgery. Terminal ileum was harvested at 6 h, 12 h, 36 h, 3 days, and 7 days following surgery and RNA was isolated. Quantitative real-time RT-PCR was utilized to determine the expression of Wnt4, Wnt6, c-myc, EphB3, EphrinB1, and EphrinB2 mRNAs. Fold change between SBR and sham was determined at each time point. Statistical analysis utilized one-way ANOVA (P < 0.05). Results. SBR resulted in early increased expression of Wnt4 (1.6-fold) by 12 h, which returned to baseline by 3 days. Significantly increased expression of c-myc occurred at 36 h (1.9-fold), peaked at 3 days (3.2-fold), and returned to baseline by 7 days. EphB3 (14.3-fold), a marker of Paneth cells, and Ephrin B2 (4.9-fold), peaked at 3 days and returned to baseline by 7 days. Conclusion. Massive intestinal resection results in early increased expression of Wnts, followed by increases in their downstream targets regulating proliferation (c-myc) and differentiation (EphB3) and cell migration (Ephrin B2). Early activation of the Wnt β-catenin pathway may be responsible for initiating intestinal adaptation.}, number={2}, journal={Journal of Surgical Research}, publisher={Elsevier BV}, author={Helmrath, M.A. and Fong, J.J. and Dekaney, C.M. and Henning, H.S.}, year={2004}, month={Oct}, pages={309–310} }