Background and AimsHuman 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.MethodsEpithelial 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.ResultsThe 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.ConclusionsWe 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.

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.

SUMMARY Microenvironmental factors modulating age-related DNA damage are unclear. Non-pituitary growth hormone (npGH) is induced in human colon, non-transformed human colon cells, and fibroblasts, and in 3-dimensional intestinal organoids with age-associated DNA damage. Autocrine/paracrine npGH suppresses p53 and attenuates DNA damage response (DDR) by inducing TRIM29 and reducing ATM phosphorylation, leading to reduced DNA repair and DNA damage accumulation. Organoids cultured up to 4 months exhibit aging markers, p16, and SA-β-galactosidase and decreased telomere length, as well as DNA damage accumulation, with increased npGH, suppressed p53, and attenuated DDR. Suppressing GH in aged organoids increases p53 and decreases DNA damage. WT mice exhibit age-dependent colon DNA damage accumulation, while in aged mice devoid of colon GH signaling, DNA damage remains low, with elevated p53. As age-associated npGH induction enables a pro-proliferative microenvironment, abrogating npGH signaling could be targeted as anti-aging therapy by impeding DNA damage and age-related pathologies.

Background 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. One potential avenue that would permit a personalized approach is to utilize human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs). iPSCs can be generated from any individual, faithfully recapitulate the genetics of the host and can be directed to form HIOs that contain both epithelial and mesenchymal cells. Our goal was to determine the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. Methods iPSCs from two control individuals and two very early onset-IBD (VEOIBD) patients with stricturing complications were obtained and directed to form HIOs. Given HIOs are heterogeneous in terms of size, shape and ratio of mesenchymal to epithelial cells, they were firstly dissociated to a single cell suspension and EpCAM was used to positively select for epithelial cells using magnetic activated cellular sorting. These EpCAM+ cells were then seeded onto transwells and EpCAM- cells were seeded as monolayers in 10% serum containing media. Both cell types were treated with the profibrotic cytokine TGFβ, and changes in the expression of selected genes were analyzed. Results iPSCs from all 4 individuals could be directed to form HIOs containing both epithelial (E-cadherin+) and mesenchymal (vimentin+) cells (see Fig. 1). In the TGFβ-treated mesenchymal cell population, expression of N-cadherin and Col1a1 was significantly increased in all four lines after 8 and 48hrs respectively, with the highest increase occurring in cells derived from VEOIBD patient 2 (see Table 1). In the TGFβ-treated epithelial cell population, Col1a1 and fibronectin expression was increased in all lines after 96hrs with the highest fold change occurring in cells derived from VEOIBD patient 1 (fibronectin) and 2 (Col1a1). Conclusion We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. We show that iPSCs generated from all selected individuals could be directed to form HIOs and that responses to the profibrotic cytokine TGFβ can be examined in both intestinal epithelial and mesenchymal cells. 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 which may ultimately lead to personalized treatments. Fluorescent images of human intestinal organoids generated from A) Control 1, B) Control 2, C) VEOIBD patient 1 and D) VEOIBD patient 2 that were immunostained with vimentin (green), E-cadherin (red) and counterstainied with DAPI (blue). All images X20