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.

Organoid-basedin vitrosystems to modelCryptosporidium parvuminfection in 2D and 3D

“…We confirmed the presence of enteroendocrine cells, goblet cells, Paneth cells, enterocytes, and stem cells by gene and protein expression, which aligns with reported data 12,18,20–22 . The presence of tuft cells and M cells was not confirmed here, but other groups have demonstrated them after adding media supplements to promote their differentiation 23,24 . We have observed cell turnover in both formats resembling cell turnover found in the gut in vivo , which occurs about every 3 days in the mouse intestine (3-5 in human intestine) 4,19,25–27 .…”

Human intestinal organoids: Modeling gastrointestinal physiology and immunopathology — current applications and limitations

“…occludin, claudins, ZO-1), which are frequently measured in organoids [14,44,[55][56][57][58]. Although these markers are useful, the gold standard for barrier assessment is transepithelial electrical resistance (TEER) in organoid monolayers or the application of fluorescent probe permeability assays in organoids [13,57].…”

“…Multiple studies using a broad range of readouts (TEER, permeability assays, and barrier function markers) on control/IBD organoids and organoid monolayers subjected to inflammatory stimuli (e.g. IFN-γ and TNF-α) or lipopolysaccharide [LPS]) consistently demonstrate reduced barrier function compared with untreated samples [13,15,57,58]. Notably, the loss of barrier function caused by inflammatory disease persists after culture, with UC colonoid monolayers exhibiting diminished baseline barrier function compared with controls [3].…”

Recent Advances in Cell‐Based In Vitro Models to Recreate Human Intestinal Inflammation

“…[204] In contrast, other studies reported having difficulties in generating long-term cultures with organoids from active inflamed regions. [207,208] Despite being an appealing tool to study inflammatory mechanisms, organoids still have roadblocks to surpass. [169] Generally, Lgr5 + adult stem cell-derived organoids only comprise the epithelia, lacking a stroma or lamina propria.…”