scholarly journals LKB1-AMPK modulates nutrient-induced changes in the mode of division of intestinal epithelial crypt cells in mice

2017 ◽  
Vol 242 (15) ◽  
pp. 1490-1498 ◽  
Author(s):  
Katherine Blackmore ◽  
Weinan Zhou ◽  
Megan J Dailey
Keyword(s):  
Stem Cell ◽  
Tissue Growth ◽  
Intestinal Epithelial ◽  
Ampk Signaling ◽  
Epithelial Stem Cell ◽  
Dividing Cells ◽  
Stem Cell Division ◽  
Induced Changes

Nutrient availability influences intestinal epithelial stem cell proliferation and tissue growth. Increases in food result in a greater number of epithelial cells, villi height and crypt depth. We investigated whether this nutrient-driven expansion of the tissue is the result of a change in the mode of intestinal epithelial stem cell division and if LKB1-AMPK signaling plays a role. We utilized in vivo and in vitro experiments to test this hypothesis. C57BL/6J mice were separated into four groups and fed varying amounts of chow for 18 h: (1) ad libitum, (2) 50% of their average daily intake (3) fasted or (4) fasted for 12 h and refed. Mice were sacrificed, intestinal sections excised and immunohistochemically processed to determine the mitotic spindle orientation. Epithelial organoids in vitro were treated with no (0 mM), low (5 mM) or high (20 mM) amounts of glucose with or without an activator (Metformin) or inhibitor (Compound C) of LKB1-AMPK signaling. Cells were then processed to determine the mode of stem cell division. Fasted mice show a greater % of asymmetrically dividing cells compared with the other feeding groups. Organoids incubated with 0 mM glucose resulted in a greater % of asymmetrically dividing cells compared with the low or high-glucose conditions. In addition, LKB1-AMPK activation attenuated the % of symmetric division normally seen in high-glucose conditions. In contrast, LKB1-AMPK inhibition attenuated the % of asymmetric division normally seen in no glucose conditions. These data suggest that nutrient availability dictates the mode of division and that LKB1-AMPK mediates this nutrient-driven effect on intestinal epithelial stem cell proliferation. Impact statement The underlying cell biology of changes in the polarity of mitotic spindles and its relevance to tissue growth is a new concept and, thus, these data provide novel findings to begin to explain how this process contributes to the regeneration and growth of tissues. We find that short-term changes in food intake in vivo or glucose availability in vitro dictate the mode of division of crypt cells. In addition, we find that LKB1-AMPK signaling modulates the glucose-induced changes in the mode of division in vitro. Identifying mechanisms involved in the mode of division may provide new targets to control tissue growth.

MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S7-S8
Author(s):  
Safina Gadeock ◽  
Cambrian Liu ◽  
Brent Polk
Keyword(s):  
Stem Cell ◽  
Stem Cell Niche ◽  
Mesenchymal Cells ◽  
Epithelial Stem Cells ◽  
Epithelial Stem Cell ◽  
Long Term Treatment ◽  
Lgr5 Expression ◽  
Cell Niche

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

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

2012 ◽  
Vol 302 (10) ◽  
pp. G1111-G1132 ◽  
Author(s):  
Laurianne Van Landeghem ◽  
M. Agostina Santoro ◽  
Adrienne E. Krebs ◽  
Amanda T. Mah ◽  
Jeffrey J. Dehmer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Populations ◽  
Intestinal Epithelial ◽  
P53 Signaling ◽  
Reporter Mice ◽  
Radiation Induced ◽  
Molecular Phenotypes ◽  
Stem Cell Populations ◽  
Induced Changes

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

scholarly journals Obesity, independent of diet, drives lasting effects on intestinal epithelial stem cell proliferation in mice

2018 ◽  
Vol 243 (10) ◽  
pp. 826-835 ◽  
Author(s):  
Weinan Zhou ◽  
Elizabeth A Davis ◽  
Megan J Dailey
Keyword(s):  
High Fat Diet ◽  
Intestinal Epithelial ◽  
Epithelial Stem Cells ◽  
High Fat ◽  
Stem Cell Proliferation ◽  
Epithelial Stem Cell ◽  
Diet Induced Obesity ◽  
And Function

The intestinal epithelium plays an essential role in nutrient absorption, hormone release, and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by intestinal epithelial stem cells located in the intestinal crypts. Obesity affects this process and results in changes in the size and function of the tissue. Because both the amount of food intake and the composition of the diet are contributing factors to developing and maintaining obesity, it is necessary to tease apart the separate contributions of obesity versus the type/amount of diet in driving the epithelial changes. C57BL/6J mice were fed a 60% high-fat diet versus a 10% low-fat diet for three months. A pair fed group was included (mice were fed with high-fat diet, but in equal kcal as that eaten by the low-fat diet- fed mice to keep them lean). We investigated the differences in (1) crypt-villus morphology in vivo, (2) the number and function of differentiated epithelial cell types in vivo, and (3) lasting effects on intestinal epithelial stem cell proliferation and growth in vitro. We found that high-fat diet-induced obesity, independent of the high-fat diet, increased crypt depth, villus height, the number of intestinal epithelial stem cells and goblet cells in vivo, and enhanced the size of the enterospheres developed from isolated IESCs in vitro. In addition, there is an interaction of obesity, type of diet, and availability of the diet (pair fed versus ad libitum) on protein and mRNA expression of alkaline phosphatase (an enzyme of enterocytes). These results suggest that high-fat diet-induced obesity, independent of the high-fat diet, induces lasting effects on intestinal epithelial stem cell proliferation, and drives the differentiation into goblet cells, but an interaction of obesity and diet drives alterations in the function of the enterocytes. Impact statement This study investigates whether obesity or the type/amount of diet differentially alters the proliferation, differentiation, growth, and function of the intestinal epithelial tissue. Although diet-induced obesity is known to alter the growth and function of the epithelium in vivo and cause lasting effects in intestinal epithelial stem cells (IESCs) in vitro, we are the first to tease apart the separate contributions of obesity versus the type/amount of diet in these processes. We found that high-fat diet (HFD)-induced obesity, independent of the HFD, drives lasting effects on IESC proliferation and differentiation into goblet cells, which may contribute to the growth of the epithelium. In addition, there is an interaction of obesity, type of diet, and availability of the diet (PF versus ad libitum) on the function of enterocytes. Identification of the factors driving the epithelial changes may provide new therapeutic strategies to control altered tissue growth and function associated with obesity.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals Regulation of sarcomagenesis by the empty spiracles homeobox genes EMX1 and EMX2

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Manuel Pedro Jimenez-García ◽  
Antonio Lucena-Cacace ◽  
Daniel Otero-Albiol ◽  
Amancio Carnero
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Neural Crest ◽  
Tumor Suppressors ◽  
Homeobox Genes ◽  
Neuronal Cell ◽  
Cell Gene Expression

AbstractThe EMX (Empty Spiracles Homeobox) genes EMX1 and EMX2 are two homeodomain gene members of the EMX family of transcription factors involved in the regulation of various biological processes, such as cell proliferation, migration, and differentiation, during brain development and neural crest migration. They play a role in the specification of positional identity, the proliferation of neural stem cells, and the differentiation of certain neuronal cell phenotypes. In general, they act as transcription factors in early embryogenesis and neuroembryogenesis from metazoans to higher vertebrates. The EMX1 and EMX2’s potential as tumor suppressor genes has been suggested in some cancers. Our work showed that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of stem cell regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES, and PROM1). EMX protein downregulation, therefore, induced the malignance and stemness of cells both in vitro and in vivo. In murine knockout (KO) models lacking Emx genes, 3MC-induced sarcomas were more aggressive and infiltrative, had a greater capacity for tumor self-renewal, and had higher stem cell gene expression and nestin expression than those in wild-type models. These results showing that EMX genes acted as stemness regulators were reproduced in different subtypes of sarcoma. Therefore, it is possible that the EMX genes could have a generalized behavior regulating proliferation of neural crest-derived progenitors. Together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-altering populations or cancer stem cells, acting as tumor suppressors in sarcoma.

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