establishment-human-epithelial-enteroids-colonoids-from-whole-tissue

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Cincinnati Children's Hospital Medical Center

Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies.

Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions.

The enteric nervous system (ENS) and its major component, enteric glial cells (EGCs), have recently been identified as a major regulator of intestinal epithelial barrier functions. Indeed, EGCs inhibit intestinal epithelial cell (IEC) proliferation and increase barrier resistance and IEC adhesion via the release of EGC-derived soluble factors. Interestingly, EGC regulation of intestinal epithelial barrier functions is reminiscent of previously reported peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARgamma ligand, 15-deoxy-(12,14)-prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARgamma dependent pathway. First, we demonstrated that the lipocalin but not the haematopoetic form for prostaglandin D synthase (PGDS), the enzyme responsible of 15dPGJ2 synthesis, was expressed in EGCs of the human submucosal plexus and of the subepithelium, as well as in rat primary culture of ENS and EGC lines. Next, 15dPGJ2 was identified in EGC supernatants of various EGC lines. 15dPGJ2 reproduced EGC inhibitory effects upon IEC proliferation, and inhibition of lipocalin PGDS expression by shRNA abrogated these effects. Furthermore, EGCs induced nuclear translocation of PPARgamma in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARgamma antagonist GW9662. Finally, EGC induced differentiation-related gene expression in IEC through a PPARgamma-dependent pathway. Our results identified 15dPGJ2 as a novel glial-derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARgamma. They also suggest that alterations of glial PGDS expression may modify intestinal epithelial barrier functions and be involved in the development of pathologies such as cancer or inflammatory bowel diseases.

Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-12,14-prostaglandin J2.

Enteric glial cells (EGCs) are important regulators of intestinal epithelial barrier (IEB) functions. EGC-derived S-nitrosoglutathione (GSNO) has been shown to regulate IEB permeability. Whether EGCs and GSNO protect the IEB during infectious insult by pathogens such as Shigella flexneri is not known.

Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione.

Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process.

Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF.

Increasing evidence suggests that enteric glial cells (EGCs) are critical for enteric neuron survival and functions. In particular, EGCs exert direct neuroprotective effects mediated in part by the release of glutathione. However, other glial factors such as those identified as regulating the intestinal epithelial barrier and in particular the omega-6 fatty acid derivative 15-deoxy-Δ¹²,¹⁴-prostaglandin J2 (15d-PGJ2) could also be involved in EGC-mediated neuroprotection. Therefore, our study aimed to assess the putative role of EGC-derived 15d-PGJ2 in their neuroprotective effects. We first showed that pretreatment of primary cultures of enteric nervous system(ENS)or humann euroblastoma cells (SH-SY5Y)with 15d-PGJ2 dose dependently prevented hydrogen peroxide neurotoxicity. Furthermore, neuroprotective effects of EGCs were significantly inhibited following genetic invalidation in EGCs of the key enzyme involved in 15d-PGJ2 synthesis, i.e. L-PGDS. We next showed that 15d-PGJ2 effects were mediated by an Nrf2 dependent pathway but were not blocked by PPARγ inhibitor (GW9662) in SH-SY5Y cells and enteric neurons. Finally, 15d-PGJ2 induced a significant increase in glutamate cysteine ligase expression and intracellular glutathione in SH cells and enteric neurons. In conclusion, we identified 15d-PGJ2 as a novel glial-derived molecule with neuroprotective effects in the ENS. This study further supports the concept that omega-6 derivatives such as 15d-PGJ2 might be used in preventive and/or therapeutic strategies for the treatment of enteric neuropathies.

The omega-6 fatty acid derivative 15-deoxy-Δ¹²,¹⁴-prostaglandin J2 is involved in neuroprotection by enteric glial cells against oxidative stress.

The monolayer of columnar epithelial cells lining the gastrointestinal tract--the intestinal epithelial barrier (IEB)--is the largest exchange surface between the body and the external environment. The permeability of the IEB has a central role in the regulation of fluid and nutrient intake as well as in the control of the passage of pathogens. The functions of the IEB are highly regulated by luminal as well as internal components, such as bacteria or immune cells, respectively. Evidence indicates that two cell types of the enteric nervous system (ENS), namely enteric neurons and enteric glial cells, are potent modulators of IEB functions, giving rise to the novel concept of a digestive 'neuronal-glial-epithelial unit' akin to the neuronal-glial-endothelial unit in the brain. In this Review, we summarize findings demonstrating that the ENS is a key regulator of IEB function and is actively involved in pathologies associated with altered barrier function.

The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease.

Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.

Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such, they have significant therapeutic potential. However, whether ISCs can survive tissue storage is unknown. We hypothesize that, although the majority of epithelial cells might die, ISCs would remain viable for at least 24 h at 4 °C. To explore this hypothesis, jejuna of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate-buffered saline at 4 °C. Delayed isolation of epithelium was performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact for 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retained high integrity for 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Cultured isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80 %. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated in human tissue, storage at 4 °C might offer a valuable temporal window for the harvesting of crypts or ISCs for therapeutic application.

Intestinal stem cells remain viable after prolonged tissue storage.

The intestinal epithelium constitutes a system of constant and rapid renewal triggered by proliferation of intestinal stem cells (ISCs), and is an ideal system for studying cell proliferation, migration and differentiation. Primary cell cultures have proved to be promising to unravel the mechanisms involved in the epithelium homeostasis. In 2009, Sato et al. established a long-term primary culture to generate epithelial organoids (enteroids) with crypt- and villus-like epithelial domains representing the complete census of progenitors and differentiated cells. Similarly, isolated ISCs expressing Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) could generate enteroids. Here, we describe methods to establish gastric, small intestinal, and colonic epithelial organoids (Basic Protocol 1) and the generation of Lgr5(+ve) single cell-derived epithelial organoids (Basic Protocol 2). We also describe the imaging techniques used to characterize those organoids (Basic Protocol 3). This in vitro model constitutes a powerful tool for studying stem cell biology and intestinal epithelial cell physiology throughout the digestive tract.

Establishment of gastrointestinal epithelial organoids.

Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.

An in vivo model of human small intestine using pluripotent stem cells.

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylorithat was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation.

Article	Year
Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions. BMC genomics\| PubMed ID: 19883504	2009
Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-12,14-prostaglandin J2. The Journal of physiology\| PubMed ID: 20478974	2010
Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione. Gut\| PubMed ID: 21139062	2011
Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 21350188	2011
The omega-6 fatty acid derivative 15-deoxy-Δ¹²,¹⁴-prostaglandin J2 is involved in neuroprotection by enteric glial cells against oxidative stress. The Journal of physiology\| PubMed ID: 22473776	2012
The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease. Nature reviews. Gastroenterology & hepatology\| PubMed ID: 23165236	2013
Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay. Gastroenterology\| PubMed ID: 23644405	2013
Intestinal stem cells remain viable after prolonged tissue storage. Cell and tissue research\| PubMed ID: 23820734	2013
Establishment of gastrointestinal epithelial organoids. Current protocols in mouse biology\| PubMed ID: 25105065	2013
An in vivo model of human small intestine using pluripotent stem cells. Nature medicine\| PubMed ID: 25326803	2014
CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation. PLoS pathogens\| PubMed ID: 25658601	2015

Maxime M. Mahe

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