Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium's role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell-derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli. Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate-dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function. The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability. We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

Growth hormone (GH) decreases with age, and GH therapy has been advocated by some to sustain lean muscle mass and vigor in aging patients and advocated by athletes to enhance performance. Environmental insults and aging lead to DNA damage, which — if unrepaired — results in chromosomal instability and tumorigenesis. We show that GH suppresses epithelial DNA damage repair and blocks ataxia telangiectasia mutated (ATM) kinase autophosphorylation with decreased activity. Decreased phosphorylation of ATM target proteins p53, checkpoint kinase 2 (Chk2), and histone 2A variant led to decreased DNA repair by nonhomologous end-joining. In vivo, prolonged high GH levels resulted in a 60% increase in unrepaired colon epithelial DNA damage. GH suppression of ATM was mediated by induced tripartite motif containing protein 29 (TRIM29) and attenuated tat interacting protein 60 kDa (Tip60). By contrast, DNA repair was increased in human nontumorous colon cells (hNCC) where GH receptor (GHR) was stably suppressed and in colon tissue derived from GHR ^–/– mice. hNCC treated with etoposide and GH showed enhanced transformation, as evidenced by increased growth in soft agar. In mice bearing human colon GH-secreting xenografts, metastatic lesions were increased. The results elucidate a mechanism underlying GH-activated epithelial cell transformation and highlight an adverse risk for inappropriate adult GH treatment.

Objective: Genome-wide association studies have identified multiple Crohn's disease (CD) susceptibility loci, including association with non-coding intergenic single-nucleotide polymorphisms (SNPs) at 10q21. Design: To fine-map the 10q21 locus, the authors genotyped 86 SNPs in 1632 CD cases and 961 controls and performed single-marker and conditional analyses using logistic regression. Results: Association with CD risk spanning 11 SNPs (p<0.001) was observed. The most significant association observed was at the non-synonymous SNP, rs7076156 (Ala62Thr), in ZNF365 . The alanine allele was over-represented in CD (p=5.23×10 ⁻⁷ ; OR=1.39 (95% CI 1.22 to 1.58)); allele frequency of 76% in CD and 69.7% in controls). Conditional analysis on rs7076156 nullified all other significant associations, suggesting that this is the causative variant at this locus. Four isoforms of ZNF365 have previously been identified, and rs7076156 is located in an exon unique to ZNF365 isoform D. The authors demonstrated, using reverse transcription-PCR, expression of ZNF365D in intestinal resections from both CD subjects and controls. Markedly reduced mean expression levels of ZNF365D were identified in Epstein–Barr virus-transformed lymphoblastoid cell lines from CD subjects homozygous for the risk allele (Ala). A whole-genome microarray expression study further suggested that the Ala62Thr change in ZNF365 isoform D is related to differential expression of the genes ARL4A , MKKS , RRAGD , SUMF2 , TDR1 and ZNF148 in CD. Conclusions: Collectively, these data support the hypothesis that the non-synonymous Ala62Thr SNP, rs7076156, underlies the association between 10q21 and CD risk and suggest that this SNP acts by altering expression of genes under the control of ZNF365 isoform D.

Background: Intestinal 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. Methods: iPSCs 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. Results: In 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. Conclusions: We 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.

Human intestinal organoids (HIOs) are increasingly being used to model intestinal responses to various stimuli, yet few studies have confirmed the fidelity of this modeling system. Given that the interferon-gamma (IFN-γ) response has been well characterized in various other cell types, our goal was to characterize the response to IFN-γ in HIOs derived from induced pluripotent stem cells (iPSCs). To achieve this, iPSCs were directed to form HIOs and subsequently treated with IFN-γ. Our results demonstrate that IFN-γ phosphorylates STAT1 but has little effect on the expression or localization of tight and adherens junction proteins in HIOs. However, transcriptomic profiling by microarray revealed numerous upregulated genes such as IDO1, GBP1, CXCL9, CXCL10 and CXCL11, which have previously been shown to be upregulated in other cell types in response to IFN-γ. Notably, “Response to Interferon Gamma” was determined to be one of the most significantly upregulated gene sets in IFN-γ-treated HIOs using gene set enrichment analysis. Interestingly, similar genes and pathways were upregulated in publicly available datasets contrasting the gene expression of in vivo biopsy tissue from patients with IBD against healthy controls. These data confirm that the iPSC-derived HIO modeling system represents an appropriate platform to evaluate the effects of various stimuli and specific environmental factors responsible for the alterations in the intestinal epithelium seen in various gastrointestinal conditions such as inflammatory bowel disease.