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Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically. Organoid cultures have been developed from multiple tissues, opening new possibilities for regenerative medicine. Here the authors demonstrate the derivation of GMP-compliant hydrogels from decellularized porcine small intestine which support formation and growth of human gastric, liver, pancreatic and small intestinal organoids.

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children 1 . The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence 2 . Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy. In a first step toward developing autologous tissue grafts for the treatment of children with intestinal failure, patient-derived jejunal organoids seeded on scaffolds of decellularized human intestinal matrix formed grafts that had jejunal properties and formed luminal structures when transplanted into mice.

The intestinal epithelium comprises a monolayer of polarised columnar cells organised along the crypt-villus axis. Intestinal stem cells reside at the base of crypts and are constantly nourished by their surrounding niche for maintenance, self-renewal, and differentiation. The cellular microenvironment including the adjacent Paneth cells, stromal cells, smooth muscle cells, and neural cells as well as the extracellular matrix together constitute the intestinal stem cell niche. A dynamic regulatory network exists among the epithelium, stromal cells, and the matrix via complex signal transduction to maintain tissue homeostasis. Dysregulation of these biological or mechanical signals could potentially lead to intestinal injury and disease. In this review, we discuss the role of different intestinal stem cell niche components and dissect the interaction between dynamic matrix factors and regulatory signalling during intestinal stem cell homeostasis.

Tissue engineering is an interdisciplinary field that combines stem cells and matrices to form functional constructs that can be used to repair damaged tissues or regenerate whole organs. Tissue stem cells can be expanded and functionally differentiated to form ‘mini-organs’ resembling native tissue architecture and function. The choice of the scaffold is also pivotal to successful tissue reconstruction. Scaffolds may be broadly classified into synthetic or biological depending upon the purpose of the engineered organ. Bioengineered intestinal grafts represent a potential source of transplantable tissue for patients with intestinal failure, a condition resulting from extensive anatomical and functional loss of small intestine and therefore digestive and absorptive capacity. Prior strategies in intestinal bioengineering have predominantly used either murine or pluripotent cells and synthetic or decellularized rodent scaffolds, thus limiting their translation. Microscale models of human intestinal epithelium on shaped hydrogels and synthetic scaffolds are more physiological, but their regenerative potential is limited by scale. Here we present a protocol for bioengineering human intestinal grafts using patient-derived materials in a bioreactor culture system. This includes the isolation, expansion and biobanking of patient-derived intestinal organoids and fibroblasts, the generation of decellularized human intestinal scaffolds from native human tissue and providing a system for recellularization to form transplantable grafts. The duration of this protocol is 12 weeks, and it can be completed by scientists with prior experience of organoid culture. The resulting engineered mucosal grafts comprise physiological intestinal epithelium, matrix and surrounding niche, offering a valuable tool for both regenerative medicine and the study of human gastrointestinal diseases. The authors present a protocol for bioengineering human intestinal mucosal grafts. This includes the isolation, expansion and biobanking of patient-derived intestinal organoids and fibroblasts and the decellularization and recellularization of human intestinal scaffolds.

BackgroundFaecal immunochemical testing (FIT) is recommended by the National Institute for Health and Care Excellence to triage symptomatic primary care patients who have unexplained symptoms but do not meet the criteria for a suspected lower gastrointestinal cancer pathway. During the COVID-19 pandemic, FIT was used to triage patients referred with urgent 2-week wait (2ww) cancer referrals instead of a direct-to-test strategy. FIT-negative patients were assessed and safety netted in a FIT negative clinic.MethodsWe reviewed case notes for 622 patients referred on a 2ww pathway and seen in a FIT negative clinic between June 2020 and April 2021 in a tertiary care hospital. We collected information on demographics, indication for referral, dates for referral, clinic visit, investigations and long-term outcomes.ResultsThe average age of the patients was 71.5 years with 54% female, and a median follow-up of 2.5 years. Indications for referrals included: anaemia (11%), iron deficiency (24%), weight loss (9%), bleeding per rectum (5%) and change in bowel habits (61%). Of the cases, 28% (95% CI 24% to 31%) had endoscopic (15%, 95% CI 12% to 18%) and/or radiological (20%, 95% CI 17% to 23%) investigations requested after clinic review, and among those investigated, malignancy rate was 1.7%, with rectosigmoid neuroendocrine tumour, oesophageal cancer and lung adenocarcinoma.ConclusionA FIT negative clinic provides a safety net for patients with unexplained symptoms but low risk of colorectal cancer. These real-world data demonstrate significantly reduced demand on endoscopy and radiology services for FIT-negative patients referred via the 2ww pathway.

Despite major advances in spatial RNA sequencing, the ability to extract temporal information in RNA sequencing experiments is still limited. Here, we describe Transcriptome Timestamping (T2), a system which harnesses naturally occurring A-to-I editing of RNA transcripts in unmodified human cells to infer transcriptional history. T2 provides age estimates for individual RNA transcripts, and serves as an endogenous molecular recorder, differentiating between complex transcriptional programs. We show that T2 can identify transient and transitional transcriptional programs in primary differentiating monocytes that are not apparent from gene expression analysis alone, including a regulatory module in the monocyte-to-macrophage transition that, to our knowledge, has not yet been described in humans. Finally, we show that T2 can also be applied to single cell data, allowing us to identify transcriptional programs in heterogeneous populations, such as asynchronously dividing cells. T2 is a scalable approach to temporal transcriptomics that can be applied to track the activity of thousands of genes in unmodified, primary human cells and tissues, with no genetic engineering.Competing Interest StatementA.G., J.B., A.W., D.M., G.Y. and S.G.R. are listed as inventors on UK patent application GB2400892.2. A.P.C. is a cofounder of Caeruleus Genomics Ltd and is an inventor on several patents related to sequencing technologies filed by Oxford University Innovations.

Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), has debilitating long-term effects on infants and children with this condition. Priority of care is to increase the child's length of functional intestine, jejunum in particular, to improve nutritional independence. Here we report a robust protocol for reconstruction of autologous intestinal mucosal grafts using primary IF patient materials. Human jejunal intestinal organoids derived from paediatric IF patients can be expanded efficiently in vitro with region-specific markers preserved after long-term culture. Decellularized human intestinal matrix with intact ultrastructure is used as biological scaffolds. Proteomic and Raman spectroscopic analyses reveal highly analogous biochemical composition of decellularized human SI and colon matrix, implying that they can both be utilised as scaffolds for jejunal graft reconstruction. Indeed, seeding of primary human jejunal organoids to either SI or colonic scaffolds in vitro can efficiently reconstruct functional jejunal grafts with persistent disaccharidase activity as early as 4 days after seeding, which can further survive and mature after transplantation in vivo. Our findings pave the way towards regenerative medicine for IF patients.