Intestinal Stem Cell Growth and Differentiation on a Tubular Scaffold with Evaluation in Small and Large Animals

Shaffiey, Shahab; Jia, Hongpeng; Keane, Timothy J.; Costello, Cait M.; Wasserman, Deena; Quidgley, Maria; Dziki, Jenna L.; Badylak, Stephen F.; Sodhi, Chhinder P.; March, John C.; Hackam, David J.

doi:10.2217/rme.15.70

Cited by 88 publications

(106 citation statements)

References 35 publications

(42 reference statements)

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“…Human fetal intestinal crypts were isolated and cultured using the protocol originally established in ref. 10 with slight modifications for human tissue (15). Human fetal tissue from less than 24-week gestation was obtained from the University of Pittsburgh Health Sciences Tissue Bank through an honest broker system after approval from the University of Pittsburgh Institutional Review Board and in accordance with the University of Pittsburgh anatomical tissue procurement guidelines.…”

Section: Methodsmentioning

confidence: 99%

“…Human fetal tissue from less than 24-week gestation was obtained from the University of Pittsburgh Health Sciences Tissue Bank through an honest broker system after approval from the University of Pittsburgh Institutional Review Board and in accordance with the University of Pittsburgh anatomical tissue procurement guidelines. Approximately 100 isolated crypts were plated in each well of a 48-well plate onto a thin layer of Matrigel (Corning) and were grown in crypt culture media comprised of Advanced DMEM/F12 (Invitrogen) with 20% (vol/vol) HyClone ES Screened FBS (Fisher), 1% Penicillin/Streptomycin (Invitrogen), 1% L-glutamine, Gentamycin, 0.2% Amphotericin B, 1% N-acetylcysteine (100 mM; Sigma), 1% N-2 supplement (100×; Invitrogen), 2% (vol/vol) B27 supplement (50×; Invitrogen), Gibco Hepes (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, 0.05 mM; Invitrogen), and ROCK Inhibitor Y-27632 (1 mM, 100×; Sigma) and supplemented with the following growth factors for the remainder of the respective experiments, with media changes occurring every 48 hours: 100 ng/mL WNT3a (Fisher), 500 ng/mL R-spondin (R&D), 100 ng/mL Noggin (Peprotech), and 50 ng/mL EGF (Fisher) (15,16). For image-based applications, enteroids were plated onto Matrigel in eight-well chamber slides (Nuc LabTek-II).…”

Section: Methodsmentioning

confidence: 99%

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Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner

Drummond

Bolock

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

134

173

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Enteroviruses are among the most common viral infectious agents of humans and are primarily transmitted by the fecal-oral route. However, the events associated with enterovirus infections of the human gastrointestinal tract remain largely unknown. Here, we used stem cell-derived enteroids from human small intestines to study enterovirus infections of the intestinal epithelium. We found that enteroids were susceptible to infection by diverse enteroviruses, including echovirus 11 (E11), coxsackievirus B (CVB), and enterovirus 71 (EV71), and that contrary to an immortalized intestinal cell line, enteroids induced antiviral and inflammatory signaling pathways in response to infection in a virus-specific manner. Furthermore, using the Notch inhibitor dibenzazepine (DBZ) to drive cellular differentiation into secretory cell lineages, we show that although goblet cells resist E11 infection, enteroendocrine cells are permissive, suggesting that enteroviruses infect specific cell populations in the human intestine. Taken together, our studies provide insights into enterovirus infections of the human intestine, which could lead to the identification of novel therapeutic targets and/or strategies to prevent or treat infections by these highly clinically relevant viruses.E nteroviruses are significant sources of human infections worldwide and are primarily transmitted by the fecal-oral route. Nonpoliovirus enteroviruses include coxsackievirus, echovirus, enterovirus 71 (EV71), and enterovirus D68 (EV-D68) and are small (∼30 nm) single-stranded RNA viruses belonging to the Picornaviridae family. In many cases, enterovirus infections remain asymptomatic, whereas in others, infection is associated with mild flu-like symptoms or much more severe outcomes such as type I diabetes, encephalomyelitis, encephalitis, myocarditis, dilated cardiomyopathy, pleurodynia, acute flaccid paralysis, or even death.The human gastrointestinal (GI) tract is a complex organ, with an epithelial surface that must provide a protective and immunological barrier in a complex and diverse microbial environment. The epithelium of the small intestine contains at least seven distinct cell subtypes that are responsible for the physiological functions of the intestine, which include nutrient absorption and defense against pathogens. The lack of models that recapitulate the complexity of the GI tract has hindered studies into many aspects of enterovirus infection in this specialized environment. Although murine models have been developed for the study of enterovirusinduced disease (1-4), many of these models require intraperitoneal (i.p.) infection, thereby bypassing the GI tract, or require ablation of the host innate immune system (5, 6). Coupled with species differences between humans and mice, there remains a need to develop human-based platforms to model enterovirus infections of the GI tract.The full repertoire of mature cells in the small intestine in vivo includes those of absorptive (enterocytes) and secretory (Paneth, goblet, and enteroendocrine) lin...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner

Drummond

Bolock

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

134

173

View full text Add to dashboard Cite

show abstract

“…Larger models of the intestinal epithelium range from air-liquid interface cultures with stroma (Ootani et al, 2009), over cells seeded on biological or synthetic scaffolds (Shaffiey et al, 2016;Torashima et al, 2016) to complex tissue-engineered intestines and living bioreactors (Levin et al, 2013;Grant et al, 2015;Cromeens et al, 2016). Progress in this area has indeed been remarkable, but all cited methods are laborious and technically difficult to perform in laboratories that lack specific tissue-engineering expertise.…”

Section: Intestinal Organoids Contain Lgr5mentioning

confidence: 99%

Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels

et al. 2017

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Multiple recent examples highlight how stem cells can self-organize in vitro to establish organoids that closely resemble their in vivo counterparts. Single Lgr5 + mouse intestinal stem cells can be cultured under defined conditions forming ever-expanding epithelial organoids that retain cell polarization, cell type diversity and anatomical organization of the in vivo epithelium. Although exhibiting a remarkable level of self-organization, the so called 'mini-guts' have a closed cystic structure of microscopic size. Here, we describe a simple protocol to generate macroscopic intestinal tubes from small cystic organoids. Embedding proliferating organoids within a contracting floating collagen gel allows them to align and fuse to generate macroscopic hollow structures ('tubes') that are lined with a simple epithelium containing all major cell types (including functional stem cells) of the small intestine. Cells lining the central contiguous lumen closely resemble the epithelial cells on luminal villi in vivo, whereas buds that protrude from the main tube into the surrounding matrix closely resemble crypts. Thus, the remarkable self-organizing properties of Lgr5 + stem cells extend beyond the level of the microscopic cystic organoid to the next, macroscopic, level of tube formation.

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“…Natural biological systems are capable of sensing environmental conditions and responding to them on multiple scales, from single cells that move along chemotactic gradients to trees that grow around boulders. One way to take advantage of these natural morphogenic systems is by directing them using external factors, such as a silken canopy "grown" by silkworms crawling along a scaffolding (Oxman et al 2014) or a functional large intestine created by seeding a collagen scaffold with intestinal progenitor cells (Shaffiey et al 2016). …”

Section: Frontier Two: Synthetic Biomaterials and Programmable Mattermentioning

confidence: 99%

Synthetic Morphogenesis

Teague¹,

Guye

Weiss

2016

Cold Spring Harb Perspect Biol

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Throughout biology, function is intimately linked with form. Across scales ranging from subcellular to multiorganismal, the identity and organization of a biological structure's subunits dictate its properties. The field of molecular morphogenesis has traditionally been concerned with describing these links, decoding the molecular mechanisms that give rise to the shape and structure of cells, tissues, organs, and organisms. Recent advances in synthetic biology promise unprecedented control over these molecular mechanisms; this opens the path to not just probing morphogenesis but directing it. This review explores several frontiers in the nascent field of synthetic morphogenesis, including programmable tissues and organs, synthetic biomaterials and programmable matter, and engineering complex morphogenic systems de novo. We will discuss each frontier's objectives, current approaches, constraints and challenges, and future potential.

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Intestinal Stem Cell Growth and Differentiation on a Tubular Scaffold with Evaluation in Small and Large Animals

Cited by 88 publications

References 35 publications

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner

Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels

Synthetic Morphogenesis

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