Adult Intestine Research Articles

Tuft cells act as regenerative stem cells in the human intestine

In mice, intestinal tuft cells have been described as a long-lived, postmitotic cell type. Two distinct subsets have been identified: tuft-1 and tuft-2 (ref. 1). By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase on interleukin-4 (IL-4) and IL-13 exposure, as reported previously in mice2–4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

A spatial expression atlas of the adult human proximal small intestine.

The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.

Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid β-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid β-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid β-oxidation.

National adult small intestine transplant (NASIT) forum: a unique, multidisciplinary team meeting for patients being considered for intestinal transplant in the UK

National adult small intestine transplant (NASIT) forum: a unique, multidisciplinary team meeting for patients being considered for intestinal transplant in the UK

The sex of organ geometry

Organs have a distinctive yet often overlooked spatial arrangement in the body1–5. We propose that there is a logic to the shape of an organ and its proximity to its neighbours. Here, by using volumetric scans of many Drosophila melanogaster flies, we develop methods to quantify three-dimensional features of organ shape, position and interindividual variability. We find that both the shapes of organs and their relative arrangement are consistent yet differ between the sexes, and identify unexpected interorgan adjacencies and left–right organ asymmetries. Focusing on the intestine, which traverses the entire body, we investigate how sex differences in three-dimensional organ geometry arise. The configuration of the adult intestine is only partially determined by physical constraints imposed by adjacent organs; its sex-specific shape is actively maintained by mechanochemical crosstalk between gut muscles and vascular-like trachea. Indeed, sex-biased expression of a muscle-derived fibroblast growth factor-like ligand renders trachea sexually dimorphic. In turn, tracheal branches hold gut loops together into a male or female shape, with physiological consequences. Interorgan geometry represents a previously unrecognized level of biological complexity which might enable or confine communication across organs and could help explain sex or species differences in organ function.

Intestinal Stem Cells Remodel in Response to Acute Notch Inhibition and Paneth Cell Loss

INTRODUCTION: Intestinal epithelial cells are rapidly renewed throughout life by intestinal stem cells (ISCs). ISC function is regulated by niche factors that orchestrate cellular proliferation and differentiation. Of these, Notch signaling is essential for ISC self-renewal and regulates the cell fate choice between absorptive cells (enterocyte) vs secretory (Paneth, goblet, enteroendocrine, and tuft) cells. Yet, the Notch niche is not fully defined. In the mature small intestine, Paneth cells neighbor each ISC and act as Notch niche cells, providing the Notch signal by expressing the Notch ligands DLL1 and DLL4 on their surface. However, studies in mice have shown that ISCs can function in the immature intestine before Paneth cells are formed, and when Paneth cells are depleted in the adult intestine. We previously observed that acute Notch inhibition via the inhibitor dibenzazepine (DBZ) resulted in Paneth cell apoptosis with intact ISCs displaying transient dysfunction. Here, we aimed to define the Notch niche after Paneth cell apoptosis in adult mice and in the developing intestine. We hypothesized that during acute Notch inhibition, ISCs remodel to compensate for Paneth cell loss by activating Notch ligand expression, and that this remodeling mimics the state of immature ISCs prior to Paneth cell emergence. METHODS: We isolated Lgr5+ ISCs by FACS from Lgr5-GFP-CreERT2 mice 24 hours after DBZ or vehicle treatment and performed bulk RNAseq analysis. To define the Notch niche during development, we analyzed postnatal intestines from Notch ligand reporter mice (Dll1-mCherry and Dll4-mCherry) crossed with the ISC reporter mice (Lgr5-GFP-CreERT2). RESULTS: ISCs isolated from Paneth cell-depleted intestines upregulated expression of Notch ligands Dll4 and Dll4 and various secretory cell markers, with expected downregulation of Notch target genes, but no alteration of other stem cell genes. This suggests that after acute Notch inhibition, Lgr5+ ISCs maintain partial stem cell identity while acquiring a signature resembling that of secretory progenitors. In the immature intestine, we showed by immunohistology that sporadic cells expressing secretory cell markers (LYZ, MUC2, and CHGA) localized to the intervillus region and co-stained with Dll1- or Dll4-mCherry+. This suggests that multiple differentiated cell types could provide the Notch signal to immature ISCs. We also observed occasional Lgr5-GFP+ ISCs that were co-positive for Dll1-mCherry, suggesting that immature ISCs can express Notch ligands at homeostasis. Further experimentation will determine whether these Dll1+ ISCs are similar to the remodeled adult ISCs after Paneth cell depletion. CONCLUSIONS: Acute Notch inhibition directly remodels ISCs to express the Notch ligands, Dll1 and Dll4 in the adult intestine. In the immature intestine, multiple secretory cell types appear to comprise the immature Notch niche. This project was funded by NIH R01 DK118023 awarded to Dr. Linda Samuelson. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Four phospholipase A2 genes encoded in the western flower thrips genome and their functional differentiation in mediating development and immunity

Eicosanoids are synthesized from phospholipids by the catalytic activity of phospholipase A2 (PLA2). Even though several PLA2s are encoded in the genome of different insect species, their physiological functions are not clearly discriminated. This study identified four PLA2 genes encoded in the western flower thrips, Frankliniella occidentalis. Two PLA2s (Fo-PLA2C and Fo-PLA2D) are predicted to be secretory while the other two PLA2s (Fo-PLA2A and Fo-PLA2B) are intracellular. All four PLA2 genes were expressed in all developmental stages, of which Fo-PLA2B and Fo-PLA2C were highly expressed in larvae while Fo-PLA2A and Fo-PLA2D were highly expressed in adults. Their expressions in different tissues were also detected by fluorescence in situ hybridization. All four PLA2s were detected in the larval and adult intestines and the ovary. Feeding double-stranded RNAs specific to the PLA2 genes specifically suppressed the target transcript levels. Individual RNA interference (RNAi) treatments led to significant developmental retardation, especially in the treatments specific to Fo-PLA2B and Fo-PLA2D. The RNAi treatments also showed that Fo-PLA2B and Fo-PLA2C expressions were required for the induction of immune-associated genes, while Fo-PLA2A and Fo-PLA2D expressions were required for ovary development. These results suggest that four PLA2s are associated with different physiological processes by their unique catalytic activities and expression patterns.

A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes

BackgroundThe enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics. New methodDevelopment of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation. ResultsMyenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells. Comparison with existing methodsThis novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials. ConclusionOur method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.

The bile salt/phospholipid ratio determines the extent of in vitro intestinal lipolysis of triglycerides: Interfacial and emulsion studies

This study focused on the protein-stabilised triglyceride (TG)/water interfaces and oil-in-water emulsions, and explored the influence of varying molar ratios of bile salts (BSs) and phospholipids (PLs) on the intestinal lipolysis of TGs. The presence of these two major groups of biosurfactants delivered with human bile to the physiological environment of intestinal digestion was replicated in our experiments by using mixtures of individual BSs and PLs under in vitro small intestinal lipolysis conditions. Conducted initially, retrospective analysis of available scientific literature revealed that an average molar ratio of 9:4 for BSs to PLs (BS/PL) can be considered physiological in the postprandial adult human small intestine. Our experimental data showed that combining BSs and PLs synergistically enhanced interfacial activity, substantially reducing oil–water interfacial tension (IFT) during interfacial lipolysis experiments with pancreatic lipase, especially at the BS/PL–9:4 ratio. Other BS/PL molar proportions (BS/PL–6.5:6.5 and BS/PL–4:9) and an equimolar amount of BSs (BS–13) followed in IFT reduction efficiency, while using PLs alone as biosurfactants was the least efficient. In the following emulsion lipolysis experiments, BS/PL–9:4 outperformed other BS/PL mixtures in terms of enhancing the TG digestion extent. The degree of TG conversion and the desorption efficiency of interfacial material post-lipolysis correlated directly with the BS/PL ratio, decreasing as the PL proportion increased. In conclusion, this study highlights the crucial role of biliary PLs, alongside BSs, in replicating the physiological function of bile in intestinal lipolysis of emulsified TGs. Our results showed different contributions of PLs and BSs to lipolysis, strongly suggesting that any future in vitro studies aiming to simulate the human digestion conditions should take into account the impact of biliary PLs – not just BSs – to accurately mimic the physiological role of bile in intestinal lipolysis. This is particularly crucial given the fact that existing in vitro digestion protocols typically focus solely on applying specific concentrations and/or compositions of BSs to simulate the action of human bile during intestinal digestion, while overlooking the presence and concentration of biliary PLs under physiological gut conditions.

Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research

BackgroundHuman induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged.MethodsTo solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids.ResultsWe established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org.ConclusionsWe have successfully improved the function and convenience of ELCs by utilizing organoid technology.

The emergence of circadian timekeeping in the intestine.

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

A host-microbial metabolite interaction gut-on-a-chip model of the adult human intestine demonstrates beneficial effects upon inulin treatment of gut microbiome.

Background: The gut and its microbiome have a major impact on many aspects of health and are therefore also an attractive target for drug- or food-based therapies. Here, we report on the added value of combining a microbiome screening model, the i-screen, with fresh intestinal tissue explants in a microfluidic gut-on-a-chip model, the Intestinal Explant Barrier Chip (IEBC). Methods: Adult human gut microbiome (fecal pool of 6 healthy donors) was cultured anaerobically in the i-screen platform for 24 h, without and with exposure to 4 mg/mL inulin. The i-screen cell-free culture supernatant was subsequently applied to the luminal side of adult human colon tissue explants (n = 3 donors), fixed in the IEBC, for 24 h and effects were evaluated. Results: The supplementation of the media with inulin promoted the growth of Anaerostipes, Bifidobacterium, Blautia, and Collinsella in the in vitro i-screen, and triggered an elevated production of butyrate by the microbiota. Human colon tissue exposed to inulin-treated i-screen cell-free culture supernatant or control i-screen cell-free culture supernatant with added short-chain fatty acids (SCFAs) showed improved tissue barrier integrity measured by a 28.2%-34.2% reduction in FITC-dextran 4000 (FD4) leakage and 1.3 times lower transport of antipyrine. Furthermore, the release of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α was reduced under these circumstances. Gene expression profiles confirmed these findings, but showed more profound effects for inulin-treated supernatant compared to SCFA-supplemented supernatant. Conclusion: The combination of i-screen and IEBC facilitates the study of complex intestinal processes such as host-microbial metabolite interaction and gut health.

Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

Direct therapeutic effect of sulfadoxine-pyrimethamine on nutritional deficiency-induced enteric dysfunction in a human Intestine Chip

Sulfadoxine-pyrimethamine (SP) antimalarial therapy has been suggested to potentially increase the birth weight of infants in pregnant women in sub-Saharan Africa, independently of malarial infection. Here, we utilized female intestinal organoid-derived cells cultured within microfluidic Organ Chips to investigate whether SP could directly impact intestinal function and thereby improve the absorption of essential fats and nutrients crucial for fetal growth. Using a human organ-on-a-chip model, we replicated the adult female intestine with patient organoid-derived duodenal epithelial cells interfaced with human intestinal endothelial cells. Nutrient-deficient (ND) medium was perfused to simulate malnutrition, resulting in the appearance of enteric dysfunction indicators such as villus blunting, reduced mucus production, impaired nutrient absorption, and increased inflammatory cytokine secretion. SP was administered to these chips in the presence or absence of human peripheral blood mononuclear cells (PBMCs). Our findings revealed that SP treatment effectively reversed multiple intestinal absorptive abnormalities observed in malnourished female Intestine Chips, as validated by transcriptomic and proteomic analyses. SP also reduced the production of inflammatory cytokines and suppressed the recruitment of PBMCs in ND chips. Our results indicate that SP could potentially increase birth weights by preventing enteric dysfunction and suppressing intestinal inflammation. This underscores the potential of SP as a targeted intervention to improve maternal absorption, subsequently contributing to healthier fetal growth. While SP treatment shows promise in addressing malabsorption issues that can influence infant birth weight, we did not model pregnancy in our chips, and thus its usefulness for treatment of malnourished pregnant women requires further investigation through clinical trials. The Bill and Melinda Gates Foundation, and the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the HDDC Organoid Core of the P30 DK034854.

RET Signaling Persists in the Adult Intestine and Stimulates Motility by Limiting PYY Release From Enteroendocrine Cells

RET tyrosine kinase is necessary for enteric nervous system development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, patients with HSCR often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility. RetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET regulates gut motility invivo, genetic, and pharmacologic approaches were used to disrupt RET in all RET-expressing cells, a subset of enteric neurons, or intestinal epithelial cells. Distinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the epithelium, rather than in enteric neurons, slowed GI motility selectively in male mice. RET kinase inhibition phenocopied this effect. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin or L-cells that release peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), both of which can alter motility. RET kinase inhibition exaggerated PYY and GLP-1 release in a nutrient-dependent manner without altering serotonin secretion in mice and human organoids. PYY receptor blockade rescued dysmotility in mice lacking epithelial RET. RET signaling normally limits nutrient-dependent peptide release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction.

Single-cell resolution of the adult zebrafish intestine under conventional conditions and in response to an acute Vibrio cholerae infection

Vibrio cholerae is an aquatic bacterium that causes severe and potentially deadly diarrheal disease. Despite the impact on global health, our understanding of host mucosal responses to Vibrio remains limited, highlighting a knowledge gap critical for the development of effective prevention and treatment strategies. Using a natural infection model, we combine physiological and single-cell transcriptomic studies to characterize conventionally reared adult zebrafish guts and guts challenged with Vibrio. We demonstrate that Vibrio causes a mild mucosal immune response characterized by Tcell activation and enhanced antigen capture; Vibrio suppresses host interferon signaling; and ectopic activation of interferon alters the course of infection. We show that the adult zebrafish gut shares similarities with mammalian counterparts, including the presence of Best4+ cells, tuft cells, and a population of basal cycling cells. These findings provide important insights into host-pathogen interactions and emphasize the utility of zebrafish as a natural model of Vibrio infection.

PTEN and soluble epoxide hydrolase in intestinal cell differentiation

Intestinal epithelial differentiation is a highly organised process. It is influenced by a variety of signalling pathways and enzymes, such as the PI3K pathway and soluble epoxide hydrolase (sEH) from arachidonic acid metabolism. We investigated the changes in the expression of enzymes and lipid messenger from the PI3K pathway, including PTEN, during intestinal cell differentiation in vitro using HT-29 and Caco2 cells and compared them with immunohistochemical patterns of these proteins in human colon. To investigate the possible crosstalk between the PI3K pathway and sEH, we treated HT-29 and Caco2 cells with the sEH inhibitor TPPU. Administration of TPPU to differentiated cells decreased the expression of PTEN, thus reversing the change in its expression observed during cell differentiation. In addition, multiplex immunofluorescence staining confirmed the relationship between the expression of PTEN and villin, a marker of intestinal cell differentiation, ranging from a moderate correlation in undifferentiated cells to a very strong correlation in differentiated cells treated with TPPU. Furthermore, we confirm that PTEN and sEH mirrored their expression patterns in samples of prenatal and adult human intestine compared to tumours using immunohistochemical staining. Taken together, it appears that PTEN and sEH cooperate in the process of intestinal cell differentiation. A better understanding of the crosstalk between the PI3K pathway and sEH and its consequences for cell differentiation is highly desirable, as several sEH inhibitors are under clinical investigation for the treatment of various diseases.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Establishment of human intestinal organoids derived from commercially available cryopreserved intestinal epithelium and evaluation for pharmacokinetic study

Human intestinal organoids (HIOs) have been reported to exert their functions in a way that mimics living organs, and HIOs-derived monolayers are expected to be applied to in vitro intestinal pharmacokinetic studies. However, HIOs are established from human tissue, which raises issues of availability and ethics. In the present study, to solve these problems, we have established intestinal organoids using commercially available cryopreserved human intestinal epithelial cells (C–IOs), and compared their functions with biopsy-derived human intestinal organoids (B–IOs) from a pharmacokinetic point of view. Both C–IOs and B–IOs reproduced the morphological features of the intestinal tract and were shown to be composed of epithelial cells. Monolayers generated from C–IOs and B–IOs (C-IO-2D, B-IO-2D, respectively) structurally mimic the small intestine. The C–IOs showed gene expression levels comparable to those of the B–IOs, which were close to those of adult human small intestine. Importantly, the C–IOs-2D showed levels of pharmacokinetics-related protein expression and activity—including cytochrome P450 3A4 (CYP3A4) and carboxylesterase 2 (CES2) enzymatic activities and P-glycoprotein (P-gp) transporter activities —similar to those of B–IOs-2D. This study addresses the difficulties associated with B–IOs and provides fundamental characteristics for the application of C–IOs in pharmacokinetic studies.

Integrin α7β1 represses intestinal absorptive cell differentiation

Intestinal epithelial cell differentiation is a highly controlled and orderly process occurring in the crypt so that cells migrating out to cover the villi are already fully functional. Absorptive cell precursors, which originate from the stem cell population located in the lower third of the crypt, are subject to several cycles of amplification in the transit amplifying (TA) zone, before reaching the terminal differentiation compartment located in the upper third. There is a large body of evidence that absorptive cell differentiation is halted in the TA zone through various epigenetic, transcriptional and intracellular signalling events or mechanisms allowing the transient expansion of this cell population but how these mechanisms are themself regulated remains obscure. One clue can be found in the epithelial cell-matrix microenvironment located all along the crypt-villus axis. Indeed, a previous study from our group revealed that α5-subunit containing laminins such as lamimin-511 and 512 inhibit early stages of differentiation in Caco-2/15 cells. Among potential receptors for laminin 511/512 is the integrin α7β1, which has previously been reported to be expressed in the human intestinal crypts and in early stages of Caco-2/15 cell differentiation. In this study, the effects of knocking down ITGA7 in Caco-2/15 cells were studied using shRNA and CRISPR/Cas9 strategies. Abolition of the α7 integrin subunit resulted in a significant increase in the level of differentiation and polarization markers as well as the morphological features of intestinal cells. Activities of focal adhesion kinase and Src kinase were both reduced in α7-knockdown cells while the three major intestinal pro-differentiation factors CDX2, HNFα1 and HNF4α were overexpressed. Two epigenetic events associated with intestinal differentiation, the reduction of tri-methylated lysine 27 on histone H3 and the increase of acetylation of histone H4 were also observed in α7-knockdown cells. On the other hand, the ablation of α7 had no effect on cell proliferation. In conclusion, these data indicate that integrin α7β1 acts as a major repressor of absorptive cell terminal differentiation in the Caco-2/15 cell model and suggest that the laminin-α7β1 integrin interaction occurring in the transit amplifying zone of the adult intestine is involved in the transient halting of absorptive cell terminal differentiation.