Development of a Human Intestinal Organoid Model for In Vitro Studies on Gut Inflammation and Fibrosis

Abstract

Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells’ fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

The Probiotic Strains Bifidοbacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii Regulate Wound Healing and Chemokine Responses in Human Intestinal Subepithelial Myofibroblasts

“…It is also worth mentioning that, when studying the effects of probiotics, mostly cell lines and animal models are used. The use of primary human cells in the current study provides more individualized and less normalized results without the need to endanger patients, while the recruitment of human colonoids and human intestinal organoids [ 58 ], which consist of both the mesenchymal and epithelial structures of the gut [ 48 ], could further enrich these results.…”

“…cSEMF production of the chemokines CXCL1, CXCL8, CXCL10 and CCL2 was measured using the Human DuoSet ® ELISAs (R&D Systems, Minneapolis, MN, USA) kit according to manufacturer’s instructions as previously described [ 48 ]. In short, cSEMFs were cultured in 6-well plates until 95% confluence, starved for 24 h and then stimulated with 10 2 or 10 4 cfu/mL of the probiotics (mix or each strain alone) for 48 h, as already described, and their supernatants were collected.…”

“…Colonic subepithelial myofibroblasts (cSEMFs) are stellate-shaped cells, residing within the lamina propria of the gut and expressing α-smooth muscle actin and vimentin [ 46 , 47 ]. cSEMFs are key components of intestinal integrity and wound healing, due to their ability to migrate to the trauma region, excrete extracellular matrix proteins, such as collagen, and support the epithelial regeneration [ 47 , 48 , 49 ]. In addition, these cells both express a wide range of cytokine receptors and produce immunological mediators and growth factors, for instance TGF-β, and are therefore involved not only in wound re-epithelization but also in the inflammatory and fibrotic cascades of the gut [ 47 , 50 , 51 ].…”

Induced Pluripotent Stem Cell-Derived Organoids: Their Implication in COVID-19 Modeling

“…Later on, further advancements in differentiation techniques and cultivation enabled the generation of iPSC-derived region-specific intestinal organoids with the integrated enteric nervous system and intestinal vasculature [ 90 , 91 , 92 ]. In the past decade, these small and large intestine organoids (enteroids and colonoids) have provided worthwhile model systems for understanding intestinal pathologies, such as colorectal cancer [ 93 ], inflammatory bowel disease [ 94 , 95 , 96 ], Crohn’s disease [ 97 , 98 ], Hirschsprung disease [ 99 ], and ulcerative colitis [ 100 ]. Similarly, intestinal organoids are powerful models used to simulate the occurrence of GIT disease mediated by viral infections, such as SARS-CoV-2 [ 86 , 87 , 101 , 102 ].…”

The application of intestinal organoids and their co-culture systems in the study of gastrointestinal diseases

“…Intestinal organoids were first established from the culture of small intestinal crypts or single ISCs (adult stem cells) in a complex extracellular matrix (ECM) by Sato et al [10]. Moreover, intestinal organoids were later generated from inducible pluripotent stem cells (iPSCs) or the human embryonic stem cell line H1 [14,15]. Intestinal organoids derived from adult stem cells and iPSCs have advantages and limitations; therefore, the selection of an intestinal organoid model depends on the purpose of the study [15].…”