INtestinal Tissue ENgineering Solution

1) Short Bowel Syndrome (SBS) is a condition that occurs when part or the entire small intestine is missing or has been removed during surgery. This condition renders the bowel incapable of fulfilling its nutritional function (intestinal failure).

2) SBS is a chronically debilitating disorder without a cure. Intravenous feeding offers a survival rate of 70% at 5 years in newborn infants. However, in the most severe cases, when only 10% of expected intestinal length is present, 5-year survival is reduced to around 20%. In particular we will focus on developing an INtestinal Tissue ENgineering Solution (INTENS) for children with SBS.

3) The objective of this programme is to deliver a functional bowel reconstruction to patients with SBS through an autologous tissue engineering strategy, overcoming the shortage of organs, and avoiding the need for immunosuppression. The work is designed to lead directly to a clinical trial for the application of the optimal protocol for tissue-engineered intestine.

WP 1
We have developed guidelines for quality assessment of intestinal cell lineages utilised for transplantation (D1.3) which were also published in the lead stem cell journal in the world (https://doi.org/10.1016/j.stem.2019.04.018) We have established protocols for quality assurance of intestinal epithelial cells (D1.4). This now means that WP1 is successfully completed.

WP 2
We designed a chemically-defined low-defect thiol-Michael addition (LDTM) hydrogels that can be formed at low polymer content without loss of mechanical integrity. These hydrogels promote the development of patterned mouse and human intestinal organoids, the latter in the absence of any animal-derived components, and thus provide a substitute for ill-defined Matrigel for successful translation of organoid technology from the lab to the clinic.

WP 3
To optimise the repopulation of the decellularised scaffolds in vitro, we first tested seeding conditions of intestinal epithelial cells (organoids) on scaffolds under static culture using RAFT system as a surrogate step to identify the optimal seeding density. We have now managed to successfully repopulate both RAFT, human and porcine decellularised scaffolds with epithelial cells, fibroblasts and endothelial cells under both static and dynamic culture. The repopulated epithelial cells were able to form polarised monolayer with sucrose digestive enzyme expression, suggesting the presence of enterocyte differentiation. More importantly, we have optimised a protocol to turn epithelial layer to form crypt-villus structures. We are developing optimal conditions for co-culturing all cell types, both static and in bioreactor, and to testtesting the functions of the recellularised intestinal graft and developing an alternative strategy engineering mucosal sheet to bypass scaffold shortages.

WP 4
The possibility to generate human intestinal organoids, mini-organs in a dish, has opened tremendous opportunities for regenerative medicine. However, the growth of these organoids relies on the use of animal-derived (growth) factors and poorly defined 3D Matrices, excluding clinical application.
In WP4, we developed and tested the ideal culturing conditions allowing optimal, safe and good manufacturing practice (cGMP)-level expansion of human intestinal organoids. We continue working on developing GMP-ready medium and iPSCs for expansion to align to the conditions to be determined in WP5 in vivo experiments to allow for clinical translation.

WP5
By gross histology, TESI develops on a scaffold with a luminal facing epithelium and surrounding mesenchyme. The vascular supply is dually contributed by de novo vascularization from donor endothelial progenitor cells and neovascularization from the host, and although capillary formation remains attenuated and rudimentary, the lymphatic network continues to grow. The epithelial surface can range in architecture from a flat epithelium to alternating villus and crypt-like structures similar to mature native small intestine. Immunofluorescent staining of mature markers of epithelial and mesenchymal cell types demonstrate the ability of OU to restore ISCs, secretory and absorptive epithelial cell types, and numerous mesenchymal support cells. In both a mouse and a swine model we can transplant intestinal stem cells.

WP 6
-Dynamic public-facing website developed, updated and integrated with eurostemcell.org.
-Development and dissemination of digital engagement tools.
-Built capacity within the INTENS consortia for public engagement through establishing a network of communicators, the production of tools to communicate digitally and through face-to-face engagement.
-Online Q&A with patient family support group.
-INTENS researchers received training at Hydra Summer School on Stem Cells and Regenerative Medicine.
-Updated “Short Bowel Syndrome – how can stem cells help?” fact sheet in line with the project developments.
-Ran an online engagement event with SBS patients and their families to provide an update on INTENS progress.
-Participated in EuroStemCell’s 2020 Capacity building workshop focused on best practice in patient engagement
-Filed a patent application “Extracellular matrix gels, and organoid cultures comprising the same” WO2021089472A1

WP 7 and WP8
WP7 activities covered all aspects of project monitoring, reporting, financial and contractual administration in accordance with the Commission’s rules, ensuring proper communication within the consortium and implementing the project governance’s decisions.
Copies of ethics approvals and renewals were collected and checked by an Ethics External Expert (Prof. Ana Carvalho), and the related deliverable (D6.9) was submitted.
Consortium worked on an amendment, including further extension to INTENS project due to COVID-19 pandemic significantly affecting in vivo work in large animal models.

We have already reported on autologous jejunal mucosal graft engineering and self-organization of stem cells into functional organoids-on-a-chip (https://www.nature.com/articles/s41586-020-2724-8 and https://www.nature.com/articles/s41591-020-1024-z) which was published in the early P4 (Sept 2020). This period has been an incredibly challenging time globally, and has had a significant impact on many aspects, including on research activity. In INTENS consortium, this particularly affected our late-stage pre-clinical development in large animals, due to facilities closures and clinical duties taking priority. Despite this, the consortium remained active in the field of stem cell/organoids engineering and applied this technology to modelling SARS-CoV-2 in the gut (https://www.nature.com/articles/s41467-021-26762-2/) Using “lab-grown stomachs” (organoids) we demonstrated that the virus can infect gastric epithelium, which supported clinical observations in a group of paediatric patients experiencing gastrointestinal symptoms associated with COVID-19, and contribute to infection via the stomach.

Periodic Reporting for period 4 - INTENS (INtestinal Tissue ENgineering Solution)

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