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

Inflammatory bowel disease (IBD) is a group of debilitating gastrointestinal (GI) disorders characterized by epithelial damage and chronic inflammation. Epithelial targeting aimed to repair and protect the epithelial barrier is central to current clinical practice. In order to explore diverse epithelial targeting strategies in IBD, novel models to reproduce intestinal epithelial biology are needed. One approach is to use patient-derived organoids (PDO) and monolayers derived from tissues throughout the intestinal tract, that can largely recapitulate the original organ at genetic, phenotypic, and histologic levels. However, variability in transcriptional and functional responses to differentiation and activation stimuli has not been systematically characterized, limiting potential applications of organoid technology. In a recent paper by Jelinsky et al. molecular and functional characterization was performed.

The authors:

• Generated PDO and monolayers from ileum and colon of non-IBD control subjects and IBD donors then characterized them at the transcriptional level.
• Characterised their response to selected media (expansion medium and various differentiation media) by histology and RNA-Seq.
• Analysed technical and biological variability in response to different media.
• Examined functional responses of monolayer models to barrier-damaging cytokines and the small molecule UC therapy Janus kinase (JAK) inhibitor Tofacitinib.

Three different media compositions were used in the study:

• CNM medium to maintain progenitor populations.
• eDM medium to promote robust barrier formation and to support enterocytes and goblet cells differentiation.
• cDM medium (developed by the authors) to promote differentiation whilst retaining stem cells.

The authors characterised the PDOs after exposure to these different media:

• CNM cultured PDOs from non-IBD donor, analysed by IHC, showed staining for Ki67 (proliferation marker) but no staining for markers associated with goblet cells (MUC2 and Alcian blue), indicative of a non-differentiated state.
• eDM or cDM treated PDOs showed development of a columnar epithelial structure, reduced Ki67 staining, and positive staining for MUC2 and Alcian blue. This is consistent with eDM and cDM driving differentiation to mature human epithelial-like structures.
• RNA-seq analysis showed that PDOs differentiated in eDM were enriched for markers of enterocytes and goblet cells, with reduced expression of genes associated with stem, Paneth, cycling TA and secretory TA cells.
• PDOs differentiated with cDM had more physiological cell-type populations than those exposed to eDM, as evidenced by increased goblet and enterocyte signatures, as well as increased enteroendocrine, Paneth, and secretory TA signatures while maintaining stem and cycling TA signatures.
• Similar transcriptional profiles were obtained from IBD-PDOs.
• Principal component analysis of the transcriptome revealed that technical and biological replicates have a high level of reproducibility.

The authors also generated PDO-derived monolayers and characterised them after exposure to the three different media:

• PDOs were dissociated into single cells and plated on transwell membranes. They were either maintained in CNM or differentiated in eDM or cDM.

• Barrier formation was monitored by measuring the transepithelial electrical resistance (TEER), which increased over time indicating an increase in tight junction formation.
• Histologic analysis of CNM-cultured monolayers revealed positive staining for Ki67 and absence of MUC2 or Alcian blue staining, suggesting a non-differentiated state.
• Colon monolayers in eDM or cDM and ileum monolayers in cDM were positive for MUC2 and Alcian blue staining, suggesting goblet cell differentiation.
• Monolayers were molecularly profiled by RNA-seq.
• Differentiation of the monolayers in eDM had a more profound effect on transcription than differentiation in cDM for both colon- and ileum-derived cultures (similarly in non-IBD and IBD derived monolayers).
• Differentiation with eDM and cDM enriched signatures for enterocyte and goblet cells, as well as TA1 and TA2, and reduced signatures for cycling TA and stem cells.
• Although PDOs an PDO-Derived monolayers showed similar differentiation signatures, the general transcriptional profile was dramatically different.

The authors evaluated the possibility of using the monolayers as a tool to study Epithelial Barrier Injury:

• PDO-derived monolayers maintained in cDM medium were stimulated with IBD-relevant pro-inflammatory cytokines (IFN-γ + TNF-α + IL-1α).
• Barrier integrity was evaluated through TEER measurement, permeability to 0.4KDa Lucifer yellow and 4 and 40 KDa dextrans and cell viability assessment using CellTiter-Glo (Promega).
• The monolayers responded to treatment with reduction of TEER, increase in permeability to Lucifer yellow and dextrans as well as reduction in total number of viable cells, confirming the negative effect of the cytokines on the barrier integrity.
• To evaluate the possibility of using monolayers as a tool to study barrier protective therapies, the authors pre-treated PDO-derived monolayers with Tofacinib, an anti-inflammatory drug used to treat IBD, prior to stimulation with the cytokine cocktail.
• Tofacitinib protected the monolayers from cytokine-induced TEER loss, increased paracellular permeability and cell death.

In conclusion the study suggests the applicability of PDOs and PDO-derived monolayers in IBD drug discovery and enables further exploration of damage and repair mechanisms in these models.