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Gut, 62(7), 1012–1023.
Ziskin JL, Dunlap D, Yaylaoglu M, Fodor IK, Forrest WF, Patel R, Ge N, Hutchins GG, Pine JK, Quirke P, Koeppen H, Jubb AM (2013).
PMID: 22637696 | DOI: 10.1136/gutjnl-2011-301195.
OBJECTIVE:
Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer.
DESIGN:
19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas.
RESULTS:
Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance.
CONCLUSION:
These data suggest that 74-85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5(+)/Ascl2(+) crypt stem cells, not Bmi1(+) stem cells. However, Olfm4 was not found to be a useful marker of Lgr5(+) cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.
Poultry Science
2023 Jan 01
Cloft, S;Miska, K;Jenkins, M;Proszkowiec-Weglarz, M;Kahl, S;Wong, E;
| DOI: 10.1016/j.psj.2023.102537
Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d post-infection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.
Poultry Science
2023 Jan 01
Cloft, S;Uni, Z;Wong, E;
| DOI: 10.1016/j.psj.2023.102495
Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5 and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.
Nature communications
2022 Mar 21
Sebastian, C;Ferrer, C;Serra, M;Choi, JE;Ducano, N;Mira, A;Shah, MS;Stopka, SA;Perciaccante, AJ;Isella, C;Moya-Rull, D;Vara-Messler, M;Giordano, S;Maldi, E;Desai, N;Capen, DE;Medico, E;Cetinbas, M;Sadreyev, RI;Brown, D;Rivera, MN;Sapino, A;Breault, DT;Agar, NYR;Mostoslavsky, R;
PMID: 35314684 | DOI: 10.1038/s41467-022-29085-y
Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.
Cell Host Microbe. 2018 Dec 12.
2018 Dec 12
Lee YS, Kim TY, Kim Y, Lee SH, Kim S, Kang SW, Yang JY, Baek IJ, Sung YH, Park YY, Hwang SW, O E, Kim KS, Liu S, Kamada N, Gao N, Kweon MN.
PMID: 30543778 | DOI: 10.1016/j.chom.2018.11.002
Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/β-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-protein-coupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner.
Oncogenic BRAF, unrestrained by TGFβ-receptor signalling, drives right-sided colonic tumorigenesis
Nature communications
2021 Jun 08
Leach, JDG;Vlahov, N;Tsantoulis, P;Ridgway, RA;Flanagan, DJ;Gilroy, K;Sphyris, N;Vázquez, EG;Vincent, DF;Faller, WJ;Hodder, MC;Raven, A;Fey, S;Najumudeen, AK;Strathdee, D;Nixon, C;Hughes, M;Clark, W;Shaw, R;S:CORT consortium, ;van Hooff, SR;Huels, DJ;Medema, JP;Barry, ST;Frame, MC;Unciti-Broceta, A;Leedham, SJ;Inman, GJ;Jackstadt, R;Thompson, BJ;Campbell, AD;Tejpar, S;Sansom, OJ;
PMID: 34103493 | DOI: 10.1038/s41467-021-23717-5
Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2023 Jun 01
Schaaf, CR;Polkoff, KM;Carter, A;Stewart, AS;Sheahan, B;Freund, J;Ginzel, J;Snyder, JC;Roper, J;Piedrahita, JA;Gonzalez, LM;
PMID: 37159340 | DOI: 10.1096/fj.202300223R
Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.
Gastroenterology
2023 Mar 01
He, S;Lei, P;Kang, W;Cheung, P;Xu, T;Mana, M;Park, C;Wang, H;Imada, S;Russell, J;Wang, J;Wang, R;Zhou, Z;Chetal, K;Stas, E;Mohad, V;Bruun-Rasmussen, P;Sadreyev, R;Hodin, R;Zhang, Y;Breault, D;Camargo, F;Yilmaz, Ö;Fredberg, J;Saeidi, N;
| DOI: 10.1053/j.gastro.2023.02.030
Background & aims Fibrosis and tissue stiffening are hallmarks of the inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). Methods We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. Results We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+ proliferating cells. Conversely, cells expressing the stem cell marker, OLFM4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of OLFM4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs towards goblet cells. Furthermore, analysis of colon samples from murine colitis models and IBD patients demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. Conclusions Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.
Cell metabolism
2022 Oct 04
Yang, J;Vamvini, M;Nigro, P;Ho, LL;Galani, K;Alvarez, M;Tanigawa, Y;Renfro, A;Carbone, NP;Laakso, M;Agudelo, LZ;Pajukanta, P;Hirshman, MF;Middelbeek, RJW;Grove, K;Goodyear, LJ;Kellis, M;
PMID: 36198295 | DOI: 10.1016/j.cmet.2022.09.004
Exercise training is critical for the prevention and treatment of obesity, but its underlying mechanisms remain incompletely understood given the challenge of profiling heterogeneous effects across multiple tissues and cell types. Here, we address this challenge and opposing effects of exercise and high-fat diet (HFD)-induced obesity at single-cell resolution in subcutaneous and visceral white adipose tissue and skeletal muscle in mice with diet and exercise training interventions. We identify a prominent role of mesenchymal stem cells (MSCs) in obesity and exercise-induced tissue adaptation. Among the pathways regulated by exercise and HFD in MSCs across the three tissues, extracellular matrix remodeling and circadian rhythm are the most prominent. Inferred cell-cell interactions implicate within- and multi-tissue crosstalk centered around MSCs. Overall, our work reveals the intricacies and diversity of multi-tissue molecular responses to exercise and obesity and uncovers a previously underappreciated role of MSCs in tissue-specific and multi-tissue beneficial effects of exercise.
Diet-induced alteration of intestinal stem cell function underlies obesity and prediabetes in mice
Nature metabolism
2021 Sep 01
Aliluev, A;Tritschler, S;Sterr, M;Oppenländer, L;Hinterdobler, J;Greisle, T;Irmler, M;Beckers, J;Sun, N;Walch, A;Stemmer, K;Kindt, A;Krumsiek, J;Tschöp, MH;Luecken, MD;Theis, FJ;Lickert, H;Böttcher, A;
PMID: 34552271 | DOI: 10.1038/s42255-021-00458-9
Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.
Proceedings of the National Academy of Sciences, 109(2), 466–471.
Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, Su N, Luo Y, Heilshorn SC, Amieva MR, Sangiorgi E, Capecchi MR, Kuo CJ (2012).
PMID: 22190486 | DOI: 10.1073/pnas.1118857109.
The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.
Experimental & molecular medicine
2022 Dec 01
Wu, SS;Lee, H;Szép-Bakonyi, R;Colozza, G;Boese, A;Gert, KR;Hallay, N;Lee, JH;Kim, J;Zhu, Y;Linssen, MM;Pilat-Carotta, S;Hohenstein, P;Theussl, HC;Pauli, A;Koo, BK;
PMID: 36494589 | DOI: 10.1038/s12276-022-00891-0
The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.
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| Description | ||
|---|---|---|
| sense Example: Hs-LAG3-sense | Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe. | |
| Intron# Example: Mm-Htt-intron2 | Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection | |
| Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G) | A mixture of multiple probe sets targeting multiple genes or transcripts | |
| No-XSp Example: Hs-PDGFB-No-XMm | Does not cross detect with the species (Sp) | |
| XSp Example: Rn-Pde9a-XMm | designed to cross detect with the species (Sp) | |
| O# Example: Mm-Islr-O1 | Alternative design targeting different regions of the same transcript or isoforms | |
| CDS Example: Hs-SLC31A-CDS | Probe targets the protein-coding sequence only | |
| EnEm | Probe targets exons n and m | |
| En-Em | Probe targets region from exon n to exon m | |
| Retired Nomenclature | ||
| tvn Example: Hs-LEPR-tv1 | Designed to target transcript variant n | |
| ORF Example: Hs-ACVRL1-ORF | Probe targets open reading frame | |
| UTR Example: Hs-HTT-UTR-C3 | Probe targets the untranslated region (non-protein-coding region) only | |
| 5UTR Example: Hs-GNRHR-5UTR | Probe targets the 5' untranslated region only | |
| 3UTR Example: Rn-Npy1r-3UTR | Probe targets the 3' untranslated region only | |
| Pan Example: Pool | A mixture of multiple probe sets targeting multiple genes or transcripts | |
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