Intestinal Mesenchymal Cells Research Articles

Identification of Key Genes in Fetal Gut Development at Single-Cell Level by Exploiting Machine Learning Techniques.

The study of fetal gut development is critical due to its substantial influence on immediate neonatal and long-term adult health. Current research largely focuses on microbiome colonization, gut immunity, and barrier function, alongside the impact of external factors on these phenomena. Limited research has been dedicated to the categorization of developing fetal gut cells. Our study aimed to enhance our understanding of fetal gut development by employing advanced machine-learning techniques on single-cell sequencing data. This dataset consisted of 62,849 samples, each characterized by 33,694 distinct gene features. Four feature ranking algorithms were utilized to sort features according to their significance, resulting in four feature lists. Then, these lists were fed into an incremental feature selection method to extract essential genes, classification rules, and build efficient classifiers. Several important genes were recognized by multiple feature ranking algorithms, such as FGG, MDK, RBP1, RBP2, IGFBP7, and SPON2. These features were key in differentiating specific developing intestinal cells, including epithelial, immune, mesenchymal, and vasculature cells of the colon, duo jejunum, and ileum cells. The classification rules showed special gene expression patterns on some intestinal cell types and the efficient classifiers can be useful tools for identifying intestinal cells.

Subepithelial Stromal Cells: Their Roles and Interactions with Intestinal Epithelial Cells during Gut Mucosal Homeostasis and Regeneration.

Intestinal epithelial cell activities during homeostasis and regeneration are well described, but their potential interactions with stromal cells remain unresolved. Exploring the functions of these heterogeneous intestinal mesenchymal stromal cells (iMSCs) remains challenging. This difficulty is due to the lack of specific markers for most functionally homogenous subpopulations. In recent years, however, novel clustering techniques such as single-cell RNA sequencing (scRNA-seq), fluorescence-activated cell sorting (FACS), confocal microscope, and computational remodeling of intestinal anatomy have helped identify and characterize some specific iMSC subsets. These methods help researchers learn more about the localization and functions of iMSC populations during intestinal morphogenic and homeostatic conditions. Consequently, it is imperative to understand the cellular pathways that regulate their activation and how they interact with surrounding cellular components, particularly during intestinal epithelial regeneration after mucosal injury. This review provides insights into the spatial distribution and functions of identified iMSC subtypes. It focuses on their involvement in intestinal morphogenesis, homeostasis, and regeneration. We reviewed related signaling mechanisms implicated during epithelial and subepithelial stromal cell crosstalk. Future research should focus on elucidating the molecular intermediates of these regulatory pathways to open a new frontier for potential therapeutic targets that can alleviate intestinal mucosa-related injuries.

Chronic arsenic exposure affects stromal cells and signaling in the small intestine in a sex-specific manner.

Arsenic is a toxicant that is ingested through drinking water and food, exposing nearly 140 million people to levels above the 10 ppb guideline concentration. Studies have shown that arsenic affects intestinal stem cells (ISCs), but the mechanisms by which arsenic alters the formation of adult cells in the small intestine are not well understood. Signals derived from intestinal stromal cells initiate and maintain differentiation. The goal of this study is to evaluate arsenic's effect on intestinal stromal cells, including PdgfrαLo trophocytes, located proximal to the ISCs, and PdgfrαHi telocytes, located proximal to the transit-amplifying region and up the villi. Adult Sox9tm2Crm-EGFP mice were exposed to 0, 33, and 100 ppb sodium arsenite in their drinking water for 13 weeks, and sections of duodenum were examined. Flow cytometry indicated that arsenic exposure dose-responsively reduced Sox9+ epithelial cells and trended toward increased Pdgfrα+ cells. The trophocyte marker, CD81, was reduced by 10-fold and 9.0-fold in the 100 ppb exposure group in male and female mice, respectively. Additionally, a significant 2.2- to 3.1-fold increase in PdgfrαLo expression was found in male mice in trophocytes and Igfbp5+ cells. PdgfrαHi protein expression, a telocyte marker, was more prevalent along the villus/crypt structure in females, whereas Gli1 expression (telocytes) was reduced in male mice exposed to arsenic. Principle coordinate analysis confirmed the sex-dependent response to arsenic exposure, with an increase in trophocyte and decrease in telocyte marker expression observed in male mice. These results imply that arsenic alters intestinal mesenchymal cells in a sex-dependent manner.

A MODEL TO STUDY INTESTINAL FIBROSIS IN IPSC-DERIVED HUMAN EPITHELIAL AND MESENCHYMAL CELLS FROM CROHN’S DISEASE PATIENTS

Abstract INTRODUCTION Intestinal fibrosis is a serious complication of Crohn's disease (CD) and is caused by the excess deposition of extracellular matrix (ECM) proteins. Numerous cell types, including intestinal epithelial and mesenchymal cells, are implicated in this but research efforts are impaired by a lack of in vitro models. To overcome this, we have previously developed a human model of intestinal fibrosis using iPSC-derived epithelial and mesenchymal cells generated from both control and CD patients. Using this model, we aimed to further characterize the transcriptomic signature associated with the fibrotic response as well as other functional hallmarks of fibrosis. METHODS iPSCs from 19 CD patients with (10) and without (9) fibrotic complications were directed to form HIOs. As iPSC-derived HIOs contain epithelial and mesenchymal cells, the organoids were dissociated to generate purified cultures of epithelial only-HIOs (eHIOs) and intestinal mesenchymal cells (MSCs). eHIO and MSC cultures were treated with TNFα and TGFβ, alone or in combination. We evaluated the transcriptomic profile of both cell types via RNA-seq and carried out additional studies via ELISA, ECM staining and scratch assay. RESULTS RNAseq analysis on the effects of cytokine induction in eHIOs lead to an enrichment in pathways associated with inflammation and regulation of the immune response. Furthermore, eHIOs from patients with fibrotic complications showed a significant upregulation of ORM1 regardless of culture conditions, which was subsequently confirmed at the protein level indicating an intrinsic difference between both patient groups. RNAseq analysis on MSC cultures found differentially expressed genes clustered in pathways associated with ECM production and remodeling, and cell migration and proliferation. Cell migration capacity of MSCs was assessed by measuring the reduction in scratch area and under basal conditions, MSCs from the fibrotic cohort had a lower rate of cell migration (40.81%) compared to the non-fibrotic cohort (20.97%); notably, the addition of TNFα appeared to accelerate the migration capacity of MSCs in both fibrotic (14.54%) and non-fibrotic (6.56%) samples. Finally, compared to untreated cultures, MSCs treated with TNFα/TGFβ exhibited a significant increase in ECM deposition (2.64- and 2.29-fold increase for non-fibrotic and fibrotic, respectively). CONCLUSION We have demonstrated the functionality of our model of intestinal fibrosis generated from iPSC-derived epithelial and mesenchymal cells from CD patients. Furthermore, we have identified both generalized and intrinsic differences in our cohort of patients with fibrotic complications. Future experiments on the role of ORM1 in intestinal fibrosis and its effects on mesenchymal cells could provide a deeper understanding of the events leading the development of intestinal fibrosis in CD patients.

Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells

BackgroundIntestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture.MethodsWe established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies.ResultsThe proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture.ConclusionsThe high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Crosstalk between CD8+ T cells and mesenchymal stromal cells in intestine homeostasis and immunity.

The intestine represents the most complex cellular network in the whole body. It is constantly faced with multiple types of immunostimulatory agents encompassing from food antigen, gut microbiome, metabolic waste products, and dead cell debris. Within the intestine, most T cells are found in three primary compartments: the organized gut-associated lymphoid tissue, the lamina propria, and the epithelium. The well-orchestrated epithelial-immune-microbial interaction is critically important for the precise immune response. The main role of intestinal mesenchymal stromal cells is to support a structural framework within the gut wall. However, recent evidence from stromal cell studies indicates that they also possess significant immunomodulatory functions, such as maintaining intestinal tolerance via the expression of PDL1/2 and MHC-II molecules, and promoting the development of CD103+ dendritic cells, and IgA+ plasma cells, thereby enhancing intestinal homeostasis. In this review, we will summarize the current understanding of CD8+ T cells and stromal cells alongside the intestinal tract and discuss the reciprocal interactions between T subsets and mesenchymal stromal cell populations. We will focus on how the tissue residency, migration, and function of CD8+ T cells could be potentially regulated by mesenchymal stromal cell populations and explore the molecular mediators, such as TGF-β, IL-33, and MHC-II molecules that might influence these processes. Finally, we discuss the potential pathophysiological impact of such interaction in intestine hemostasis as well as diseases of inflammation, infection, and malignancies.

Administration of intestinal mesenchymal stromal cells reduces colitis-associated cancer in C57BL/6J mice modulating the immune response and gut dysbiosis

BackgroundPatients with inflammatory bowel disease (IBD) have a higher risk of developing colitis-associated colorectal cancer (CAC) with poor prognosis. IBD etiology remains undefined but involves environmental factors, genetic predisposition, microbiota imbalance (dysbiosis) and mucosal immune defects. Mesenchymal stromal cell (MSC) injections have shown good efficacy in reducing intestinal inflammation in animal and human studies. However, their effect on tumor growth in CAC and their capacity to restore gut dysbiosis are not clear. MethodsThe outcome of systemic administrations of in vitro expanded human intestinal MSCs (iMSCs) on tumor growth in vivo was evaluated using the AOM/DSS model of CAC in C57BL/6J mice. Innate and adaptive immune responses in blood, mesenteric lymph nodes (MLNs) and colonic tissue were analyzed by flow cytometry. Intestinal microbiota composition was evaluated by 16S rRNA amplicon sequencing. ResultsiMSCs significantly inhibited colitis and intestinal tumor development, reducing IL-6 and COX-2 expression, and IL-6/STAT3 and PI3K/Akt signaling. iMSCs decreased colonic immune cell infiltration, and partly restored intestinal monocyte homing and differentiation. iMSC administration increased the numbers of Tregs and IFN-γ+CD8+ T cells in the MLNs while decreasing the IL-4+Th2 response. It also ameliorated intestinal dysbiosis in CAC mice, increasing diversity and Bacillota/Bacteroidota ratio, as well as Akkermansia abundance, while reducing Alistipes and Turicibacter, genera associated with inflammation. ConclusionAdministration of iMSCs protects against CAC, ameliorating colitis and partially reverting intestinal dysbiosis, supporting the use of MSCs for the treatment of IBD.

A NEW MODEL TO STUDY INTESTINAL FIBROSIS USING IPSC-DERIVED HUMAN EPITHELIAL AND MESENCHYMAL CELLS FROM CROHN’S DISEASE PATIENTS

Intestinal fibrosis is a serious complication of Crohn’s disease (CD) and is caused by the excess deposition of extracellular matrix protein. There are no therapies to prevent or treat this and surgical intervention remains the only treatment option. Numerous cell types, including intestinal epithelial and mesenchymal cells, are implicated in this but research efforts are impaired by a lack of in vitro models. Human intestinal organoids (HIOs), derived from induced pluripotent stem cells (iPSCs), are comprised of both of these cell types; therefore iPSC-derived HIOs represent an approach in which an unlimited number of patient specific cells could be generated for such models. Our goal was to confirm the feasibility of using iPSC-derived epithelial and mesenchymal cells from CD patients to model intestinal fibrosis.

A NEW MODEL TO STUDY INTESTINAL FIBROSIS USING IPSC-DERIVED HUMAN EPITHELIAL AND MESENCHYMAL CELLS FROM CROHN’S DISEASE PATIENTS

Abstract INTRODUCTION Intestinal fibrosis is a serious complication of Crohn's disease (CD) and is caused by the excess deposition of extracellular matrix protein. There are no therapies to prevent or treat this and surgical intervention remains the only treatment option. Numerous cell types, including intestinal epithelial and mesenchymal cells, are implicated in this but research efforts are impaired by a lack of in vitro models. Human intestinal organoids (HIOs), derived from induced pluripotent stem cells (iPSCs), are comprised of both of these cell types; therefore iPSC-derived HIOs represent an approach in which an unlimited number of patient specific cells could be generated for such models. Our goal was to confirm the feasibility of using iPSC-derived epithelial and mesenchymal cells from CD patients to model intestinal fibrosis METHODS iPSCs from CD patients with and without fibrotic complications were directed to form HIOs. As iPSC-derived HIOs contain epithelial and mesenchymal cells, the organoids were dissociated to generate purified cultures of epithelial-only HIOs (eHIOs) and mesenchymal cells. Both cell types were cultured with TNFα and TGFβ, and responses were assayed after 8, 24 and 48 hrs. Further assessment on the transcriptomic profile of the cultures was conducted using the Nanostring nCounter Fibrosis Panel. RESULTS Mesenchymal cells treated with TNFα for 48 hrs showed a significant increase in MMP9 expression, whereas TGFβ elicited an upregulation in COL1A1 and COL5A1. Further analysis via Nanostring identified the upregulation of numerous other fibrosis-related genes in response to TNFα stimulation, mainly: CCL2, IL6, MMP7, and MMP9. In eHIO cultures, after 24 hrs of cytokine exposure, we detected a significant upregulation in EMT (NCAD, VIM, and SNAI2) and fibrosis genes (COL1A1, COL5A1, and MMP9), mainly in response to TGFβ and the combination TNFα/TGFβ. Nanostring further identified a total of 121 differentially expressed genes, mostly in response to these treatments, particularly: CCL2, IL11, MMP9, MMP10. Overall, Nanostring analysis found an upregulation in pathways associated with ECM production and remodeling, immunomodulation, and ligands associated with EMT and cell migration. CONCLUSION We have successfully generated a system to assay intestinal fibrosis in a patient-specific manner using iPSC-derived epithelial and mesenchymal cells from CD patients. Our model elicited a reliable and reproducible response to inflammatory and fibrogenic stimuli, measured by the differential expression of key fibrosis-associated genes. Future studies will focus on a broader analysis of these data via RNA-seq to investigate a potential fibrogenic phenotype and to identify novel genes/pathways that may play in role in the pathophysiology of intestinal fibrosis in CD.

HNF4α Acts as Upstream Functional Regulator of Intestinal Wnt3 and Paneth Cell Fate

The intestinal epithelium intrinsically renews itself exvivo via the proliferation of Lgr5+ intestinal stem cells, which is sustained by the establishment of an epithelial stem cell niche. Differentiated Paneth cells are the main source of epithelial-derived niche factor supplies and produce Wnt3 as an essential factor in supporting Lgr5+ stem cell activity in the absence of redundant mesenchymal Wnts. Maturation of Paneth cells depends on canonical Wnt signaling, but few transcriptional regulators have been identified to this end. The role of HNF4α in intestinal epithelial cell differentiation is considered redundant with its paralog HNF4γ. However, it is unclear whether HNF4α alone controls intrinsic intestinal epithelial cell growth and fate in the absence of a mesenchymal niche. We used transcriptomic analyses to dissect the role of HNF4α in the maintenance of jejunal epithelial culture when cultured exvivo as enteroids in the presence or absence of compensatory mesenchymal cells. HNF4α plays a crucial role in supporting the growth and survival of jejunal enteroids. Transcriptomic analyses revealed an autonomous function of HNF4α in Wnt3 transcriptional regulation and Paneth cell differentiation. We showed that Wnt3a supplementation or co-culture with intestinal subepithelial mesenchymal cells reversed cell death and transcriptional changes caused by the deletion of Hnf4a in jejunal enteroids. Our results support the intrinsic epithelial role of HNF4α in regulating Paneth cell homeostasis and intestinal epithelium renewal in the absence of compensatory Wnt signaling.

Immune niches orchestrated by intestinal mesenchymal stromal cells lining the crypt-villus.

The mammalian intestine is an organ that can be spatially defined by two axes: longitudinal and vertical. Such anatomical structure ensures the maintenance of a relatively immuno-quiescent and proliferation-promoting crypt for intestinal stem cell differentiation while actively warding off the invading intestinal microbes at the villus tip during digestion and nutrient absorption. Such behavior is achieved by the fine coordination among intestinal epithelial cells, intestinal mesenchymal stromal cells and tissue-resident immune cells like myeloid cells and lymphocytes. Among these cell types resided in the colon, intestinal mesenchymal stromal cells are considered to be the essential link between epithelium, vasculature, neuronal system, and hematopoietic compartment. Recent advancement of single cell and spatial transcriptomics has enabled us to characterize the spatial and functional heterogeneity of intestinal mesenchymal stromal cells. These studies reveal distinctive intestinal mesenchymal stromal cells localized in different regions of the intestine with diverse functions including but not limited to providing cytokines and growth factors essential for different immune cells and epithelial cells which predict niche formation for immune function from the villus tip to the crypt bottom. In this review, we aim to provide an overall view of the heterogeneity of intestinal mesenchymal stromal cells, the spatial distribution of these cells along with their interaction with immune cells and the potential regulatory cytokine profile of these cell types. Summarization of such information may enrich our current understanding of the immuno-regulatory functions of the newly identified mesenchymal stromal cell subsets beyond their epithelial regulatory function.

Gut homeostasis at a glance.

The intestine, a rapidly self-renewing organ, is part of the gastrointestinal system. Its major roles are to absorb food-derived nutrients and water, process waste and act as a barrier against potentially harmful substances. Here, we will give a brief overview of the primary functions of the intestine, its structure and the luminal gradients along its length. We will discuss the dynamics of the intestinal epithelium, its turnover, and the maintenance of homeostasis. Finally, we will focus on the characteristics and functions of intestinal mesenchymal and immune cells. In this Cell Science at a Glance article and the accompanying poster, we aim to present the most recent information about gut cell biology and physiology, providing a resource for further exploration.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

SummaryA major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Tissue Niches Formed by Intestinal Mesenchymal Stromal Cells in Mucosal Homeostasis and Immunity.

The gastrointestinal tract is the largest mucosal surface in our body and accommodates the majority of the total lymphocyte population. Being continuously exposed to both harmless antigens and potentially threatening pathogens, the intestinal mucosa requires the integration of multiple signals for balancing immune responses. This integration is certainly supported by tissue-resident intestinal mesenchymal cells (IMCs), yet the molecular mechanisms whereby IMCs contribute to these events remain largely undefined. Recent studies using single-cell profiling technologies indicated a previously unappreciated heterogeneity of IMCs and provided further knowledge which will help to understand dynamic interactions between IMCs and hematopoietic cells of the intestinal mucosa. In this review, we focus on recent findings on the immunological functions of IMCs: On one hand, we discuss the steady-state interactions of IMCs with epithelial cells and hematopoietic cells. On the other hand, we summarize our current knowledge about the contribution of IMCs to the development of intestinal inflammatory conditions, such as infections, inflammatory bowel disease, and fibrosis. By providing a comprehensive list of cytokines and chemokines produced by IMCs under homeostatic and inflammatory conditions, we highlight the significant immunomodulatory and tissue niche forming capacities of IMCs.

Total Flavone of Abelmoschus manihot Ameliorates TNBS-Induced Colonic Fibrosis by Regulating Th17/Treg Balance and Reducing Extracellular Matrix.

Background and Aims: Surgery remains the major available strategy in inflammatory bowel disease (IBD) fibrotic strictures because no available drugs have sufficient prevention and treatment in this complication. This study aimed to evaluate the efficacy of the total flavone of Abelmoschus manihot L. Medic (TFA) on the development of colonic fibrosis in mice and its possible mechanism. Methods: The 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced chronic colonic inflammation-associated fibrosis mice were used to evaluate anti-fibrosis of TFA using macroscopic, histological, immunohistochemical analyses, ELISA, Masson staining, Verhoeff’s von Gieson staining, transcription-quantitative polymerase chain reaction, and immunoblot analysis. Results: Oral administration of TFA attenuated body weight loss, reduced colon length shortening, lowered the morphological damage index score, and notably ameliorated the inflammatory response. TFA downregulated proinflammatory cytokines IL-6, IL-17, TNF-α, IFN-γ productions, and increased the levels of anti-inflammatory cytokine IL-10 and TGF-β. The histological severity of the colonic fibrosis was also notably improved by the TFA treatment and associated with a significant reduction in the colonic expression of col1a2, col3a2, and hydroxyproline. TFA inhibits α-SMA, TGF-β, vimentin, TIMP-1 expression, increasing MMPs, thereby inhibiting activated intestinal mesenchymal cells and extracellular matrix (ECM) deposition. Conclusion: Together, we herein provide the evidence to support that TFA may restore the imbalance of Th17/Treg and decrease the generation of ECM. This may be a potential mechanism by which TFA protects the intestine under inflammatory conditions and acts as a therapeutic agent for the treatment of intestinal fibrosis in Crohn’s disease.

Development of a Personalized Intestinal Fibrosis Model Using Human Intestinal Organoids Derived From Induced Pluripotent Stem Cells.

BackgroundIntestinal fibrosis is a serious complication of Crohn’s disease. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. Human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs) contain these cell types, and our goal was to determine the feasibility of utilizing these to develop a personalized intestinal fibrosis model.MethodsiPSCs from 2 control individuals and 2 very early onset inflammatory bowel disease patients with stricturing complications were obtained and directed to form HIOs. Purified populations of epithelial and mesenchymal cells were derived from HIOs, and both types were treated with the profibrogenic cytokine transforming growth factor β (TGFβ). Quantitative polymerase chain reaction and RNA sequencing analysis were used to assay their responses.ResultsIn iPSC-derived mesenchymal cells, there was a significant increase in the expression of profibrotic genes (Col1a1, Col5a1, and TIMP1) in response to TGFβ. RNA sequencing analysis identified further profibrotic genes and demonstrated differential responses to this cytokine in each of the 4 lines. Increases in profibrotic gene expression (Col1a1, FN, TIMP1) along with genes associated with epithelial-mesenchymal transition (vimentin and N-cadherin) were observed in TGFβ -treated epithelial cells.ConclusionsWe demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. This now permits the generation of near unlimited quantities of patient-specific cells that could be used to reveal cell- and environmental-specific mechanisms underpinning intestinal fibrosis.

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis.

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.

The Bacterial Microbiota Promotes Normal Hematopoiesis Via Interferon Alpha and NOD1 Signaling Pathways

Long-term antibiotic therapy is associated with hematological side effects such as neutropenia and anemia. Our lab and others have shown that long-term antibiotic treatment in mice leads to bone marrow suppression and agranulocytosis in mice through depletion of the commensal bacteria. Our work further showed that Stat1-deficient mice phenocopy the bone marrow suppression phenotype of antibiotic-treated mice, suggesting that commensal microbiota mediate hematopoiesis via Stat1 signaling. However, the upstream actors of this pathway and the bacterial mediators required for commensal microbiota regulation of normal hematopoiesis still remain poorly understood. Such knowledge will be essential for understanding how to treat antibiotic-associated cytopenias. We hypothesize that microbial products detected by host cells trigger STAT1 signaling to potentiate normal hematopoiesis.To identify the host cells that require Stat1 for microbiota-promoted hematopoiesis, we treated conditional Stat1 knock-out mice with two weeks of antibiotic therapy. Of the four conditional knock-out mice we evaluated (LepR-Cre, Villin-Cre, Vav-iCre, LysM-Cre), only the mice deficient in STAT1 in hematopoietic cells (Vav-iCre Stat1 fl/fl) phenocopied the bone marrow suppression of antibiotic-treated mice. Our data suggest that STAT1 signaling is necessary in non-myeloid hematopoietic cells, but not intestinal epithelial cells or mesenchymal stromal cells for microbiota-promoted hematopoiesis. Non-competitive transplantation of Stat1 -/- bone marrow into wild type mice validated these findings; mice lacking STAT1 only in hematopoietic tissues phenocopied the Vav-iCre mice, consistent with a specific role for microbiota-mediated STAT1 signaling in the hematopoietic compartment.To assess the upstream mediator of STAT1 signaling in this biological context, we treated interferon (IFN) receptor knock out mice with two weeks of antibiotics. Of the three types of IFN receptor knock-outs evaluated, only mice deficient in type I IFN signaling phenocopied the bone marrow suppression of antibiotic-treated mice. These findings suggest that type I IFN signaling, and not type II or III, was required for microbiota-dependent hematopoiesis. These results were validated by showing that the administration of pegylated-IFNα was sufficient to rescue the depletion of type I IFN-STAT1 signaling in antibiotic-treated mice.To determine the microbial signals that may potentiate hematopoiesis, we evaluated two microbial products that were previously shown to alter hematopoiesis and to activate type I IFN signaling (Iwamura et al. Blood 2017 & Steed et al. Science 2017). We discovered that oral administration of these commensal-derived products, the metabolite desaminotyrosine (DAT) or NOD1 ligand (NOD1L), a motif of peptidoglycan, were each sufficient to rescue the hematopoietic defects induced by antibiotics in mice. To test whether these products rescue hematopoiesis by activating STAT1 signaling, we attempted to rescue the hematopoietic defects in Stat1 -/- mice. These studies showed that NOD1L rescues granulocyte but not progenitor counts in Stat1 -/- mice, suggesting that NOD1 and type I IFN signaling work together at the progenitor level, but independently at the downstream myeloid progenitor level to promote granulopoiesis.Overall, our studies expand our understanding of the signaling pathways by which the microbiota promotes normal hematopoiesis and identify novel therapeutic agents that can be used to ameliorate antibiotic-induced BM suppression. DisclosuresNo relevant conflicts of interest to declare.

Intestinal mesenchymal cells regulate immune responses and promote epithelial regeneration in vitro and in dextran sulfate sodium-induced experimental colitis in mice.

Disruption of the intestinal mucosal tolerance, that is, the immunological unresponsiveness to innocuous food antigens and the commensal microbiota, in the colon is associated with several chronic diseases including inflammatory bowel disease (IBD). Understanding the mechanisms responsible for intestinal mucosal tolerance has potential translational value for its therapy and management. Human intestinal mesenchymal cells (iMCs) play important roles in colonic mucosal tolerance, but further studies on their tissue regenerative and immunomodulatory capacities are necessary in order to fully understand their function in health and disease. In this study, we have isolated and analysed the capacity of human iMCs to promote wound healing and modulate immune responses in vitro and in vivo, using the dextran sulfate sodium (DSS)-induced colitis model. Cultured iMCs were CD45- CD73+ CD90+ CD105+ and accelerated the wound closure in a normal colon mucosa (NCM) 356 human epithelial cell wound healing assay. Furthermore, iMCs blocked the LPS-mediated induction of TNF-α in THP-1 macrophages and inhibited the proliferation of peripheral blood mononuclear cells, partly through the induction of indoleamine-2,3-dioxygenase. In DSS colitic mice, iMCs administration reduced the disease activity index and ameliorated intestinal tissue damage and permeability. Furthermore, iMCs reduced intestinal inflammation, evidenced by a decreased mRNA expression of pro-inflammatory cytokines, reduced IL-1β secretion by intestinal explants and inhibited colonic iNOS protein expression. Our data show that human iMCs isolated from the noninflamed intestine possess tissue-regenerative and immunomodulatory capacities that could potentially be harnessed/restored in order to reduce IBD severity.

DEVELOPMENT OF A PERSONALIZED MODEL OF INTESTINAL FIBROSIS USING HUMAN INTESTINAL ORGANOIDS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS

Intestinal fibrosis is a serious complication of inflammatory bowel disease (IBD) with > 20% of Crohn’s disease patients developing this complication within 10 years of diagnosis. Despite improvements in anti-inflammatory medication, its incidence remains stubbornly high and thus far surgical intervention remains the only treatment option. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models.