Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping.

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.

Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.

The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.

Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium.

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."

Diabetes risk gene and Wnt effector Tcf7l2/TCF4 controls hepatic response to perinatal and adult metabolic demand.

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.

Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.

Urine-derived bladder cancer organoids (urinoids) as a tool for cancer longitudinal response monitoring and therapy adaptation.

BACKGROUND: Bladder cancer is one of the most common cancer types worldwide. Generally, research relies on invasive sampling strategies. METHODS: Here, we generate bladder cancer organoids directly from urine (urinoids). In this project, we establish 12 urinoid lines from 22 patients with non-muscle and muscle-invasive bladder tumours, with an efficiency of 55%. RESULTS: The histopathological features of the urinoids accurately resemble those of the original bladder tumours. Genetically, there is a high concordance of single nucleotide polymorphisms (92.56%) and insertions & deletions (91.54%) between urinoids and original tumours from patient 4. Furthermore, these urinoids show sensitivity to bladder cancer drugs, similar to their tissue-derived organoid counterparts. Genetic analysis of longitudinally generated tumoroids and urinoids from one patient receiving systemic immunotherapy, identify alterations that may guide the choice for second-line therapy. Successful treatment adaptation was subsequently demonstrated in the urinoid setting. CONCLUSION: Therefore, urinoids can advance precision medicine in bladder cancer as a non-invasive platform for tumour pathogenesis, longitudinal drug-response monitoring, and therapy adaptation.

Transcription factor achaete scute-like 2 controls intestinal stem cell fate.

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Canonical Wnt signaling negatively modulates regulatory T cell function.

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

Enteroendocrine and tuft cells support Lgr5 stem cells on Paneth cell depletion.

Cycling intestinal Lgr5+ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

Profiling proliferative cells and their progeny in damaged murine hearts.

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.

Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.

In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.

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, stomach and hair follicles. 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. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. 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.

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon.

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.

Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67-RFP allele.

Cycling Lgr5+ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5+ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5high stem cells are continuously in cell cycle, while a fraction of Lgr5low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5low Ki67- cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.

Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors.

LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.

Porcupine inhibitor suppresses paracrine Wnt-driven growth of Rnf43;Znrf3-mutant neoplasia.

Rnf43 (RING finger protein 43) and Znrf3 (zinc/RING finger protein 3) (RZ) are two closely related transmembrane E3 ligases, encoded by Wnt target genes, that remove surface Wnt (wingless-int) receptors. The two genes are mutated in various human cancers. Such tumors are predicted to be hypersensitive to, yet still depend on, secreted Wnts. We previously showed that mutation of RZ in the intestine yields rapidly growing adenomas containing LGR5(+) (leucine-rich repeat-containing G-protein coupled receptor 5) stem cells and Wnt3-producing Paneth cells. We now show that removal of Paneth cells by Math1 mutation inhibits RZ(-/-) tumor formation. Similarly, deletion of Wnt3 inhibits tumorigenesis. Treatment of mice carrying RZ(-/-) intestinal neoplasia with a small molecule Wnt secretion inhibitor (porcupine inhibitor C59) strongly inhibited growth, whereas adjacent normal crypts remained intact. These results establish that paracrine Wnt secretion is an essential driver of RZ(-/-) tumor growth and imply that a therapeutic window exists for the use of porcupine inhibitors for RZ-mutant cancers.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking.

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny.

Although Lgr5(+) intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5(+) intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5(+) cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.