JAK/STAT-1 Signaling Is Required for Reserve Intestinal Stem Cell Activation during Intestinal Regeneration Following Acute Inflammation

Summary The intestinal epithelium serves as an essential barrier to the outside world and is maintained by functionally distinct populations of rapidly cycling intestinal stem cells (CBC ISCs) and slowly cycling, reserve ISCs (r-ISCs). Because disruptions in the epithelial barrier can result from pathological activation of the immune system, we sought to investigate the impact of inflammation on ISC behavior during the regenerative response. In a murine model of αCD3 antibody-induced small-intestinal inflammation, r-ISCs proved highly resistant to injury, while CBC ISCs underwent apoptosis. Moreover, r-ISCs were induced to proliferate and functionally contribute to intestinal regeneration. Further analysis revealed that the inflammatory cytokines interferon gamma and tumor necrosis factor alpha led to r-ISC activation in enteroid culture, which could be blocked by the JAK/STAT inhibitor, tofacitinib. These results highlight an important role for r-ISCs in response to acute intestinal inflammation and show that JAK/STAT-1 signaling is required for the r-ISC regenerative response

Development of a Human Primary Intestine Chip and Identification of Species-Specific Microbiome Metabolites Enhancing Enterohemorrhagic E. Coli Pathogenesis

The human intestine and its microbiome play an extremely important role in regulating human pathologies such as inflammatory disease and bacterial infections. Due to the complexity of human studies there has been a great effort in developing in vitro models mimicking the complex human intestinal physiology, such as Gut Chip. The Gut Chip is a microfluidic device that emulates normal tissue-tissue interface and mimics the complex physiological, physical and biochemical environment of a living intestine. This model has been previously established utilizing cell lines, such as Caco-2 or HT-29 cells, that were isolated from tumors. These cells harbor several genetic mutations and have altered expression of some receptors for bacterial toxins (i.e. Shiga Toxin), compared to normal colonic epithelium. Therefore, these Gut Chips do not fully recapitulate normal intestinal functions and would not be applicable to the investigation of important human diseases such as bacterial infections. Thus, in this study, we developed a primary human Small Intestine Chip (Intestine Chip) containing primary endothelial cells and primary epithelial cells isolated from biopsies. Epithelial cells, harvested from intestinal tissue, are expanded as intestinal organoids, dissociated, and cultured on a extracellular-matrix-coated porous membrane, in one channel of the Intestine Chip. The channel laying on the opposite side of the porous membrane is lined with primary human endothelial cells and the Chip is kept under fluidic flow and cyclic deformation. The intestinal epithelium cultured in the Intestine Chip gives rise to villi-like projections formed by polarized epithelial cells that differentiate in several lineages similar to what occurring in intestinal organoids. However, in the Intestine Chip, the apical side of epithelial cells is exposed to an accessible lumen subject to fluidic flow, compared to the closed lumen of intestinal organoids. Transcriptomics analysis indicates that the Intestine Chip more closely mimics human duodenum in vivo compared to intestinal duodenal organoids. Thanks to the fluidic flow, the lumen of the Intestine Chip can be collected and used to measure mucus secretion, nutrients digestion, and intestinal barrier function over an extended period of time. Furthermore, presence of fluidic flow and the separation between vascular and intestinal luminal compartment allow the investigation of the effect of microbiome metabolites and intestinal luminal pathogens. Intestinal pathogens differentially affect different host species and, in some cases, commensal bacteria in a specie can be extremely harmful to a different host. Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic E. coli (EHEC) infection, while mice are relatively resistant. Studying the contribution of the microbiome to this differential susceptibility is difficult due to complex hostpathogen- microbiome interactions. Here, we optimized the Intestine Chip model to include pathogenic bacteria, microbiome metabolites and colonic epithelial cells (Colon Chip), more relevant in the context of EHEC infection. We show that epithelial injury is greater when the Colon Chip is exposed to human microbiome metabolites compared to murine. Using a multiomics approach we identified four specific human microbiome metabolites that are sufficient to mediate this effect. Thus, the greater injury seen in human could be partially explained by the presence of specific microbiome metabolites. These metabolites preferentially induce the expression of flagellin, increase bacteria motility, and worsen epithelial injury. The study described in this thesis provides a new model to investigate colonic infectious disease and the role played by microbiome metabolites. Additionally, it offers new insights in EHEC pathogenesis mediated by human microbiome metabolites, that could partially explain increased susceptibility to EHEC in certain human population, such as children. These findings also provide the basis to therapies aimed at modulating the intestinal content in order to provide protection from life-threatening pathogens

Distinct Processes and Transcriptional Targets Underlie CDX2 Requirements in Intestinal Stem Cells and Differentiated Villus Cells

Summary Lgr5-expressing intestinal stem cells (ISCs) renew the adult gut epithelium by producing mature villus cells (VCs); the transcriptional basis for ISC functions remains unclear. RNA sequencing analysis identified transcripts modulated during differentiation of Lgr5+ ISCs into VCs, with high expression of the intestine-restricted transcription factor (TF) gene Cdx2 in both populations. Cdx2-deleted mouse ISCs showed impaired proliferation and long-term inability to produce mature lineages, revealing essential ISC functions. Chromatin immunoprecipitation sequencing analysis of CDX2 in Lgr5+ ISCs, coupled with mRNA profiling of control and Cdx2−/− ISCs, identified features of CDX2 regulation distinct from VCs. Most CDX2 binding in ISCs occurs in anticipation of future gene expression, but whereas CDX2 primarily activates VC genes, direct ISC targets are activated and repressed. Diverse CDX2 requirements in stem and differentiated cells may reflect the versatility of TFs that specify a tissue in development and control the same tissue in adults

Microfluidic Organ-on-a-Chip Models of Human Intestine

Microfluidic organ-on-a-chip models of human intestine have been developed and used to study intestinal physiology and pathophysiology. In this article, we review this field and describe how microfluidic Intestine Chips offer new capabilities not possible with conventional culture systems or organoid cultures, including the ability to analyze contributions of individual cellular, chemical, and physical control parameters one-at-a-time; to coculture human intestinal cells with commensal microbiome for extended times; and to create human-relevant disease models. We also discuss potential future applications of human Intestine Chips, including how they might be used for drug development and personalized medicine

Telomerase expression marks transitional growth-associated skeletal progenitor/stem cells: mTert marks skeletal progenitor/stem cells

©2021 The Authors. Stem Cells published by Wiley Periodicals LLC on behalf of AlphaMed Press. Skeletal progenitor/stem cells (SSCs) play a critical role in postnatal bone growth and maintenance. Telomerase (Tert) activity prevents cellular senescence and is required for maintenance of stem cells in self-renewing tissues. Here we investigated the role of mTert-expressing cells in postnatal mouse long bone and found that mTert expression is enriched at the time of adolescent bone growth. mTert-GFP+ cells were identified in regions known to house SSCs, including the metaphyseal stroma, growth plate, and the bone marrow. We also show that mTert-expressing cells are a distinct SSC population with enriched colony-forming capacity and contribute to multiple mesenchymal lineages, in vitro. In contrast, in vivo lineage-tracing studies identified mTert+ cells as osteochondral progenitors and contribute to the bone-forming cell pool during endochondral bone growth with a subset persisting into adulthood. Taken together, our results show that mTert expression is temporally regulated and marks SSCs during a discrete phase of transitional growth between rapid bone growth and maintenance

The colonic epithelium plays an active role in promoting colitis by shaping the tissue cytokine profile

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines

Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids

Here we describe a method for fabricating a primary human Small Intestine-on-a-Chip (Intestine Chip) containing epithelial cells isolated from healthy regions of intestinal biopsies. The primary epithelial cells are expanded as 3D organoids, dissociated, and cultured on a porous membrane within a microfluidic device with human intestinal microvascular endothelium cultured in a parallel microchannel under flow and cyclic deformation. In the Intestine Chip, the epithelium forms villi-like projections lined by polarized epithelial cells that undergo multi-lineage differentiation similar to that of intestinal organoids, however, these cells expose their apical surfaces to an open lumen and interface with endothelium. Transcriptomic analysis also indicates that the Intestine Chip more closely mimics whole human duodenum in vivo when compared to the duodenal organoids used to create the chips. Because fluids flowing through the lumen of the Intestine Chip can be collected continuously, sequential analysis of fluid samples can be used to quantify nutrient digestion, mucus secretion and establishment of intestinal barrier function over a period of multiple days in vitro. The Intestine Chip therefore may be useful as a research tool for applications where normal intestinal function is crucial, including studies of metabolism, nutrition, infection, and drug pharmacokinetics, as well as personalized medicine.ISSN:2045-232

Dormant Intestinal Stem Cells Are Regulated by PTEN and Nutritional Status

The cellular and molecular mechanisms underlying adaptive changes to physiological stress within the intestinal epithelium remain poorly understood. Here, we show that PTEN, a negative regulator of the PI3K→AKT→mTORC1-signaling pathway, is an important regulator of dormant intestinal stem cells (d-ISCs). Acute nutrient deprivation leads to transient PTEN phosphorylation within d-ISCs and a corresponding increase in their number. This release of PTEN inhibition renders d-ISCs functionally poised to contribute to the regenerative response during re-feeding via cell-autonomous activation of the PI3K→AKT→mTORC1 pathway. Consistent with its role in mediating cell survival, PTEN is required for d-ISC maintenance at baseline, and intestines lacking PTEN have diminished regenerative capacity after irradiation. Our results highlight a PTEN-dependent mechanism for d-ISC maintenance and further demonstrate the role of d-ISCs in the intestinal response to stress

Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated by microbiome metabolites

Abstract Background Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. Results We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites—4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid—that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. Conclusion Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection

Expansion of transit amplifying cells in animals with colitis.

<p>(A) Markers of goblet cells, enterocytes (Ent.), enteroendocrine cells (EE), and stem cells measured by RNA microarray. Each gene was mean centered, and the means of inflamed and noninflamed groups are indicated in red and gray, respectively. Horizontal and vertical lines represent mean +/− standard error. Significance was determined by unpaired <i>t</i> test. * represents <i>p</i> ≤ 0.05, ** represents <i>p</i> ≤ 0.01, *** represents <i>p</i> ≤ 0.001. (B) Average fold change between inflamed and noninflamed groups for each of the genes in panel A. (C) Enrichment plots for experimentally derived gene sets for secretory progenitors (Secpro), stem cells, enterocytes (Ent), goblet cell-deficient epithelium (Atoh null), and transit-amplifying cell (TAC) epithelium. (D) Normalized enrichment scores for the gene sets indicated in panel C; false discovery rate (FDR) values are indicated above or below each bar. (E) Mean-centered expression values of the 4 chemokines found in the TAC gene set. The means of inflamed and noninflamed groups are indicated in red and gray, respectively. Horizontal and vertical lines represent mean +/− standard error. All genes showed a <i>p</i> < 0.001 in a <i>t</i> test, as indicated by ***. Underlying numerical values for panels A and C–E are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s001" target="_blank">S1 Data</a>.</p