@article{schaaf_polkoff_carter_stewart_sheahan_freund_ginzel_snyder_roper_piedrahita_et al._2023, title={A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease}, volume={37}, ISSN={["1530-6860"]}, DOI={10.1096/fj.202300223R}, abstractNote={Abstract}, number={6}, journal={FASEB JOURNAL}, author={Schaaf, Cecilia R. and Polkoff, Kathryn M. and Carter, Amber and Stewart, Amy S. and Sheahan, Breanna and Freund, John and Ginzel, Joshua and Snyder, Joshua C. and Roper, Jatin and Piedrahita, Jorge A. and et al.}, year={2023}, month={Jun} }
 @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{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={ In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs. }, 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}, 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{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} }