Molecular and Functional Characterization of Human Intestinal Organoids and Monolayers for Modeling Epithelial Barrier

Abstract

Background: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier.

Methods: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib.

Results: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib.

Conclusions: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.

Keywords: barrier function; cell differentiation; organoids; tofacitinib.

Graphical Abstract

Figure 1.

PDO respond to selected media with consistent gene expression and physiological cell-type diversity. A, PCA analysis of biological replicates of control donor-derived PDO (upper panel) and IBD-PDOs (lower panel) grown in media specified. B, Control donor-derived and IBD-PDOs differentially expressed genes. Red symbols are significantly different between conditions. C, Heat map depicting expression of epithelial related genes. D, Epithelial cell-type signatures after differentiation are represented as fold-change relative to undifferentiated.

Figure 2.

PDO-derived monolayers display consistent responses to media for gene expression and physiological cell-type signatures. A, PCA analysis of biological replicates of control donor-derived monolayers (upper panel) and IBD-PDO-derived monolayers (lower panel) grown in media specified. B, Control donor- and IBD-PDO-derived monolayers differentially expressed genes. Red symbols are significantly different between conditions. C, Heat map depicting expression of epithelial cell-type related genes. D, Epithelial cell-type signatures after differentiation are represented as fold-change relative to undifferentiated.

Figure 3.

Intestinal cells respond differently to plating in organoid vs monolayer formats. A, PCA analysis of organoid and monolayer formats. B, Genes differentially expressed between monolayers and organoids. Red symbols are significantly different between conditions (FDR < .01). C, Differentially expressed pathways between monolayers and organoids. D, Normalized expression of selected epithelial genes. Box plots denote the average and standard deviation of 3 technical replicates of 2 to 3 independent biological replicates per condition. Relevant statistics were performed using ttest with Bonferroni multiple comparison correction (*P < .05, **P < .01, ***P < .001, ****P < .0001).

Figure 4.

Intestinal monolayers can be applied to model epithelial barrier injury. A-E, Control PDO-derived monolayers were monitored for TEER before and after stimulation with cytokines listed. E, Viability and permeability relative to wells treated with staurosporine were measured at 24 hours following cytokine addition. Cultures were differentiated with cDM media on day 5 and cytokines were administered on day 10. (A-D) The concentration of cytokine indicates the amount of each individual cytokine in the cocktail. E, The “varying” cytokine concentrations in the cocktail are reflected on the x axis and “constant” cytokines were held at 5 ng/mL in the cocktail. Nonlinear regression analysis and EC50 values were calculated at 5- or 24-hours postcytokine stimulation. Points represent the average of 3 wells (A-D) or 2 wells (E), and error bars indicate the standard deviation. Relevant statistics were performed using 1-way ANOVA with Tukey’s multiple comparison test. Statistics refer to differences between cytokine treatment and no treatment groups.

Figure 5.

Monolayers derived from control and IBD-PDOs can be applied to model responses to IBD therapies. Monolayers were pretreated with various concentrations of tofacitinib 1 hour prior to stimulation with inflammatory cytokines. A, Colon and (B) ileum monolayers derived from control donors or (C and D) colon derived from 2 UC patients, or (E) ileum derived from a CD patient were monitored for changes in TEER. Nonlinear regression analysis and IC50 values were calculated at 5 or 24 hours postcytokine stimulation. Points represent the average of 3 wells and error bars indicate the standard deviation. Relevant statistics were performed using 1-way ANOVA with Tukey’s multiple comparison test. Statistics refer to differences between tofacitinib treatment and no treatment groups.