In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture

Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.

doi:10.1038/nprot.2013.153

Cited by 594 publications

(641 citation statements)

References 13 publications

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“…Because a complex set of DAF-dependent intracellular signals and cytoskeletal rearrangements is required for CVB3 infection of polarized human intestinal epithelium (10, 36), we wanted to be sure that human DAF functions to permit infection of murine cells. To test this, we made use of recent technical advances in the culture and maintenance of nontransformed primary epithelial cells (27,37,38). We isolated intestinal stem cells from the duodenum of a DAF transgenic mouse and from a wild-type littermate and used these to produce polarized epithelial cell monolayers.…”

Section: Resultsmentioning

confidence: 99%

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Pan

Zhang

Odenwald

et al. 2015

J Virol

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In vitro, infection of polarized human intestinal epithelial cells by coxsackievirus B3 (CVB3) depends on virus interaction with decay-accelerating factor (DAF), a receptor expressed on the apical cell surface. Although mice are highly susceptible to CVB3 infection when virus is delivered by intraperitoneal injection, infection by the enteral route is very inefficient. Murine DAF, unlike human DAF, does not bind virus, and we hypothesized that the absence of an accessible receptor on the intestinal surface is an important barrier to infection by the oral route. We generated transgenic mice that express human DAF specifically on intestinal epithelium and measured their susceptibility to infection by a DAF-binding CVB3 isolate. Human DAF permitted CVB3 to bind to the intestinal surface ex vivo and to infect polarized monolayers of small-intestinal epithelial cells derived from DAF transgenic mice. However, expression of human DAF did not facilitate infection by the enteral route either in immunocompetent animals or in animals deficient in the interferon alpha/beta receptor. These results indicate that the absence of an apical receptor on intestinal epithelium is not the major barrier to infection of mice by the oral route. IMPORTANCE CVB3 infection of human intestinal epithelial cells depends on DAF at the apical cell surface, and expression of human DAF on murine intestinal epithelial cells permits their infection in vitro. However, expression of human DAF on the intestinal surface of transgenic mice did not facilitate infection by the oral route. Although the role of intestinal DAF in human infection has not been directly examined, these results suggest that DAF is not the critical factor in mice.C oxsackieviruses belong to the genus Enterovirus of the family Picornaviridae, a group of small, nonenveloped viruses with a single-strand, positive-sense RNA genome (1). Group B coxsackieviruses (CVBs) are important causes of viral myocarditis and meningitis, and they have been proposed to be a possible triggering agent for juvenile diabetes (1).CVBs, like other enteroviruses, are transmitted by the fecaloral route and are believed to initiate infection by crossing the intestinal mucosa, which is lined by polarized epithelial cells with distinct apical and basolateral surfaces. In polarized epithelial cells, the primary CVB receptor, the coxsackievirus and adenovirus receptor (CAR) (2-4), is not expressed on the apical cell surface but is instead confined to the basolateral surface, where it functions as part of the intercellular tight junction (5); CAR is thus inaccessible to virus approaching the apical cell surface from the intestinal lumen. Some CVB isolates bind to a second receptor, decay-accelerating factor (DAF) (6-8). Unlike CAR, DAF is highly expressed on the apical surface of polarized epithelial cells, and infection of polarized cells depends on virus attachment to DAF (9). In addition to providing a docking site for virus, DAF mediates a series of intracellular signals that permit virus to move ac...

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Section: Resultsmentioning

confidence: 99%

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Pan

Zhang

Odenwald

et al. 2015

J Virol

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“…Caco-2 cells were grown in DMEM containing 10% FBS, 15 mM HEPES, and nonessential amino acids (pH 7.4). T84 cells were cultured in DMEM/ determine whether manipulation of this pathway in small intestinal epithelial organoids (34) and/or in stem cell-mimicking spheroids (35) results in a dramatic phenotype.…”

Section: Methodsmentioning

confidence: 99%

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis

Wu¹,

Xu²,

Lu³

et al. 2017

Journal of Clinical Investigation

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Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction-associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.

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“…Enteroid culture and assessments of proliferation activity. Small intestinal enteroid cultures from WT C57BL/6 mice were established and maintained per published protocols (9,22). Briefly, a 1-cm 2 segment of ileal tissue was removed and washed in DMEM/F12 containing 10% FBS to inactivate endogenous proteases.…”

Section: Methodsmentioning

confidence: 99%

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5⁺ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice

Riehl

Santhanam

Foster

et al. 2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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Riehl TE, Santhanam S, Foster L, Ciorba M, Stenson WF. CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5 ϩ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice. Am J Physiol Gastrointest Liver Physiol 309: G874 -G887, 2015. First published October 1, 2015; doi:10.1152/ajpgi.00123.2015.-Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 wk of age to wild-type, CD44Ϫ/Ϫ , and TLR4 Ϫ/Ϫ mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5 ϩ stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5 ϩ reporter mice from postnatal day 7 to day 14 decreased Lgr5 ϩ cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5 ϩ stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4. hyaluronic acid; CD44; Toll-like receptor 4; intestinal growth; crypt fission; Lgr5 stem cell proliferation HYALURONIC ACID (HA), a glycosaminoglycan polymer with repeating units of disaccharides composed of D-glucuronic acid and N-acetyl-D-glucosamine, is an important constituent of the extracellular matrix. HA is secreted by many cell types; it is assembled at the plasma membrane by HA synthases (HASs) and extruded into the extracellular space. The HA chain can extend up to 2 ϫ 10 5 disaccharides and up to 25 m long. HA expression is increased in many injury states, including Crohn's disease in humans, dextran sodium sulfate (DSS)-induced colitis in mice, and intestinal radiation injury (8,16,24). The angiogenic, inflammatory, and immunostimulatory effects of HA are mediated by binding to its receptors. The HA receptor CD44 is expressed on the plasma membrane of most cells, including fibroblasts, smooth muscle cells, epithelial cells, and immune cells (19). In addition to binding to CD44, HA also binds to Toll-like receptors 2 and 4 (TLR2 and TLR4), which are components of the innate immune system (7, 20, 24). TLR2 and TLR4 are widel...

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In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture

Cited by 594 publications

References 13 publications

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5⁺ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice

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In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture

Cited by 594 publications

References 13 publications

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice

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CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5⁺ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice