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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.

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet. Dietary serine and glycine starvation has emerged as a potential therapy for cancer. Here, the authors show that inhibition of PHGDH, which mediates the first step in the serine synthesis pathway, improves the therapeutic efficacy of serine depletion diet in mouse xenograft models.

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOB is ) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc , Kras G12D and Trp53 R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids. Mass cytometry in combination with a thiol-reactive barcoding strategy allows analysis and comparison of cell-type-specific signaling networks in organoids.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5⁺) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5⁺ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5⁺ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4⁺ DCS cells promote organoid formation of single Lgr5⁺ colon stem cells. DCS cells can be massively produced from Lgr5⁺ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4⁺ DCS cells serve as Paneth cell equivalents in the colon crypt niche.

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.

Short bowel syndrome (SBS), a condition defined by insufficient absorptive intestinal epithelium, is a rare disease, with an estimated prevalence up to 0.4 in 10,000 people. However, it has substantial morbidity and mortality for affected patients. The mainstay of treatment in SBS is supportive, in the form of intravenous parenteral nutrition, with the aim of achieving intestinal autonomy. The lack of a definitive curative therapy has led to attempts to harness innate developmental and regenerative mechanisms to engineer neo-intestine as an alternative approach to addressing this unmet clinical need. Exciting advances have been made in the field of intestinal tissue engineering (ITE) over the past decade, making a review in this field timely. In this Review, we discuss the latest advances in the components required to engineer intestinal grafts and summarize the progress of ITE. We also explore some key factors to consider and challenges to overcome when transitioning tissue-engineered intestine towards clinical translation, and provide the future outlook of ITE in therapeutic applications and beyond.Intestinal tissue engineering offers a potential therapeutic option for short bowel syndrome. This Review examines the progress in intestinal tissue engineering, discusses the components required for engineered intestinal grafts, preclinical progress and efforts towards clinical translation, including challenges to overcome.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages. The generation of primary organoids, from fetal fluid-derived epithelial stem or progenitor cells, offers the possibility of modeling different developing tissues during gestation, even beyond the limits of pregnancy termination.

Yap signalling is crucial for intestinal regeneration, but its role is largely dispensable in homeostasis. Two studies now reveal Yap-dependent mechanisms of intestinal regeneration and tissue organization: transient expansion of a rare cell type after damage in vivo and Notch–Dll activation for symmetry breaking in organoid development.

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

A mouse model of liver damage has identified a population of Lrg5 + liver stem cells that can generate hepatoctyes and bile ducts in vivo. Wake-up call for liver stem cells Hans Clevers and colleagues have identified a quiescent population of adult liver stem cells that can be 'woken up' by damage. In mice subject to liver damage, small cells expressing the Wnt target gene Lgr5 accumulate near the bile ducts. One of these cells was used to grow large numbers of bipotent stem cells in vitro . The stem cells were converted to functional hepatocytes in vitro , and when liver organoids were transplanted into a mouse model of tyrosinemia type I liver disease, islands of apparently normal hepatocytes appeared in the liver. Whether these hepatocytes are fully functional is not yet known, but the results are promising for regenerative approaches in the liver. The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon 1 , stomach 2 and hair follicles 3 . A three-dimensional culture system allows long-term clonal expansion of single Lgr5 + stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture 2 , 4 , 5 . A crucial component of the culture medium is the Wnt agonist RSPO1 6 , the recently discovered ligand of LGR5 7 , 8 . Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ + cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5 + cells generate hepatocytes and bile ducts in vivo . Single Lgr5 + cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah −/− mice. These findings indicate that previous observations concerning Lgr5 + stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.