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Human induced pluripotent stem cell-derived intestinal organoids (HIOs) are powerful tools to study development and diseases of the gastrointestinal tract. Here, the authors develop a directed differentiation protocol to generate mesenchyme-free HIOs that can be patterned towards proximal small intestine or colonic epithelium, and demonstrated their utility in modeling CFTR function.

Abstract Background To investigate whether alterations in the developing intestinal microbiota and immune markers precede celiac disease (CD) onset in infants at familial risk of developing the disease. Methods A nested case-control study was carried out as part of a larger prospective cohort study, which included healthy full-term newborns (> 200) with at least one first relative with biopsy-verified CD. The present study includes cases of CD (n = 10) and the best-matched controls (n = 10) who did not develop the disease after 5-year follow-up. Fecal microbiota, assessed by high-throughput 16S rRNA gene amplicon sequencing, and immune parameters were profiled at 4 and 6 months of age and related to CD onset. Results The microbiota of infants who remained healthy showed an increase in bacterial diversity over time, characterized by increases in Firmicutes families, but not those who developed CD. Infants who subsequently developed CD showed a significant reduction in sIgA levels over time, while those who remained healthy showed increases in TNF-α correlated to Bifidobacterium spp. An increased relative abundance of Bifidobacterium longum was associated with control children while increased proportions of Bifidobacterium breve and Enterococcus spp. were associated with CD development. Conclusion The findings suggest that alterations in the early trajectory of gut microbiota in infants at CD risk could influence the immune maturation process and predispose to CD, although larger population studies are warranted to confirm this hypothesis.

Abstract Multi-modal three dimensional (3D) optical imaging combining both structural sensitivity and molecular specificity is highly desirable in biomedical research. In this paper, we present a method termed oblique scanning laser microscopy (OSLM) to combine optical coherence tomography (OCT), for simultaneously volumetric structural and molecular imaging with cellular resolution in all three dimensions. Conventional 3D laser scanning fluorescence microscopy requires repeated optical sectioning to create z-stacks in depth. Here, the use of an obliquely scanning laser eliminates the z-stacking process, then allows highly efficient 3D OCT and fluorescence imaging by using only one raster scan. The current setup provides ~3.6 × 4.2 × 6.5 μm resolution in fluorescence imaging, ~7 × 7 × 3.5 μm in OCT in three dimensions, and the current speed of imaging is up to 100 frames per second (fps) over a volume about 0.8 × 1 × 0.5 mm3. We demonstrate several mechanisms for molecular imaging, including intrinsically expressed GFP fluorescence, autofluorescence from Flavin proteins, and exogenous antibody-conjugated dyes. We also demonstrate potential applications in imaging human intestinal organoids (HIOs), colon mucosa, and retina.

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

Interactions between environmental factors and predisposing genes could be involved in the development of coeliac disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated measures. In the whole population, breast-feeding promoted colonization of C. leptum group, B. longum and B. breve, while formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover, increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B. fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants' gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder.