Sabine Middendorp

A functional CFTR assay using primary cystic fibrosis intestinal organoids

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro–expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

Tissue-specific mutation accumulation in human adult stem cells during life

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Peyer's Patch M Cells Derived from Lgr5+ Stem Cells Require SpiB and Are Induced by RankL in Cultured “Miniguts”

ABSTRACT Peyers patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-called M cells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In SpiB−/− mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. We show that in intestinal organoid (“minigut”) cultures, stimulation with RankL induces SpiB expression within 24 h and expression of other M cell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells.

Adult Stem Cells in the Small Intestine Are Intrinsically Programmed with Their Location-Specific Function

Differentiation and specialization of epithelial cells in the small intestine are regulated in two ways. First, there is differentiation along the crypt‐villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine, or M cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum, and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location‐specific functional properties are intrinsically programmed within stem cells or if continuous signaling from mesenchymal cells is necessary to maintain the location‐specific identity of the small intestine. Using the pure epithelial organoid technique, we show that region‐specific gene expression profiles are conserved throughout long‐term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4‐regulated gene expression is only allowed in absence of WNT signaling. These data show that location‐specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location‐specific extracellular signals. In light of the potential future clinical application of small intestine‐derived organoids, our data imply that it is important to generate GATA4‐positive and GATA4‐negative cultures to regenerate all essential functions of the small intestine. Stem Cells 2014;32:1083–1091

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Many Inflammatory Bowel Disease Risk Loci Include Regions That Regulate Gene Expression in Immune Cells and the Intestinal Epithelium

BACKGROUND & AIMS The contribution of genetic factors to the pathogenesis of inflammatory bowel disease (IBD) has been established by twin, targeted-sequencing, and genome-wide association studies. These studies identified many risk loci, and research is underway to identify causal variants. These studies have focused mainly on protein-coding genes. We investigated other functional elements in the human genome, such as regulatory regions. METHODS Using acetylated histone 3 lysine 27 chromatin immunoprecipitation and sequencing, we identified tens of thousands of potential regulatory regions that are active in intestinal epithelium (primary intestinal crypts and cultured organoids) isolated from resected material and from biopsies collected during ileo-colonoscopies and immune cells (monocytes, macrophages, CD34(+), CD4(+), and CD8(+)). We correlated these regions with susceptibility loci for IBD. RESULTS We have generated acetylated histone 3 lysine 27 profiles from primary intestinal epithelium and cultured organoids, which we have made publically available. We found that 45 of 163 single nucleotide polymorphisms (SNPs) associated with IBD overlap specifically with active regulatory elements. In addition, by taking strong linkage disequilibrium into account, another 47 IBD-associated SNPs colocalized with active regulatory elements through other SNPs in their vicinity. Altogether, 92 of 163 IBD-associated SNPs correlated with distinct active regulatory elements-a frequency 2.5- to 3.5-fold greater than that expected from random sampling. The variations in these SNPs often create or disrupt known binding motifs; they might affect the binding of transcriptional regulators to alter expression of regulated genes. CONCLUSIONS In addition to variants in protein coding genes, variants in noncoding DNA regulatory regions that are active in intestinal epithelium and immune cells are potentially involved in the pathogenesis of IBD.

NKT cells in mucosal immunity

The gastrointestinal tract allows the residence of an almost enumerable number of bacteria. To maintain homeostasis, the mucosal immune system must remain tolerant to the commensal microbiota and eradicate pathogenic bacteria. Aberrant interactions between the mucosal immune cells and the microbiota have been implicated in the pathogenesis of inflammatory disorders, such as inflammatory bowel disease (IBD). In this review, we discuss the role of natural killer T cells (NKT cells) in intestinal immunology. NKT cells are a subset of non-conventional T cells recognizing endogenous and/or exogenous glycolipid antigens when presented by the major histocompatibility complex (MHC) class I-like antigen-presenting molecules CD1d and MR1. Upon T-cell receptor (TCR) engagement, NKT cells can rapidly produce various cytokines that have important roles in mucosal immunity. Our understanding of NKT-cell-mediated pathways including the identification of specific antigens is expanding. This knowledge will facilitate the development of NKT cell-based interventions and immune therapies for human intestinal diseases.

Human enteroids: preclinical models of non-inflammatory diarrhea

Researchers need an available and easy-to-use model of the human intestine to better understand human intestinal physiology and pathophysiology of diseases, and to offer an enhanced platform for developing drug therapy. Our work employs human enteroids derived from each of the major intestinal sections to advance understanding of several diarrheal diseases, including those caused by cholera, rotavirus and enterohemorrhagic Escherichia coli. An enteroid bank is being established to facilitate comparison of segmental, developmental, and regulatory differences in transport proteins that can influence therapy efficacy. Basic characterization of major ion transport protein expression, localization and function in the human enteroid model sets the stage to study the effects of enteric infection at the transport level, as well as to monitor potential responses to pharmacological intervention.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

Significance Microvillus inclusion disease (MVID) is a rare intestinal enteropathy resulting in severe diarrhoea in neonates. Here, we have generated an intestine-specific knockout mouse model for Myosin Vb, the gene causing MVID in the majority of human patients. Our mouse model completely recapitulates the intestinal human MVID phenotype, including severe diarrhoea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules in villus enterocytes. In addition, we identify a newly identified role of Myo5b in trafficking of basolateral proteins, in the apical localization of the brush border membrane fusion protein syntaxin 3 (STX3), and in early differentiation of enterocytes. Our data indicate a role of MYO5B in regulating polarity of epithelial cells and have important implications for future treatment options for MVID patients. Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.

Naive T cells in the gut of newly diagnosed, untreated adult patients with inflammatory bowel disease.

Background:The phenotype of the T-cell subpopulations and their related cytokine networks in the gastrointestinal mucosa of patients with inflammatory bowel disease can potentially be used as a predictive value for clinical course and response to therapy. Here, we analyzed T-cell subpopulations in newly diagnosed, untreated adult patients and correlated them with clinical presentation. Methods:Mucosal biopsies from duodenum, ileum, and colon mucosa of patients with Crohns disease and ulcerative colitis and controls were obtained. The simple endoscopy score in Crohns disease and the full Mayo score in ulcerative colitis were used to score disease activity. Mucosa-infiltrating T cells were characterized by flow cytometric immunophenotyping and were stimulated to assess cytokine secretion. Results:Based on the expression of the maturation and activation markers CD45RA and CD27, we identified 4 different profiles. Profile A contained mainly CD45RA+CD27+ naive T cells; profile B contained mainly CD45RA−CD27+ central memory T cells; profile C contained mainly CD45RA−CD27− effector memory T cells; and profile D consisted of similar percentages of these aforementioned subpopulations. Profile A was only observed in the ileum/colon of patients with inflammatory bowel disease, associated with upper gastrointestinal location and perianal disease in Crohns disease and expressed more tumor necrosis factor &agr; and less interferon &ggr;. In contrast, profile D was restricted to controls. There was no correlation between the different T-cell profiles and endoscopic disease activity. Conclusions:Newly diagnosed patients with inflammatory bowel disease display different T-cell maturation profiles in the gut mucosa, corresponding to distinct cytokine responses. Follow-up studies are needed to determine whether the profiles associate with clinical course and response to therapy.