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Key Points Intestinal stem cells (ISCs) are not static entities but are instead involved in many dynamical processes. ISCs are equipotent and continuously replace each other in neutral events. The ISC phenotype is the sum of all markers and features that are commonly associated with stem cells in the intestine. Therefore, the ISC phenotype is continuously changing as new markers and features are being identified. ISC activity is the ability of cells to initiate clonal long-term, multipotent lineages and is typically assessed by lineage tracing experiments. ISC potential refers to the display of ISC activity solely in a specific context but not during homeostasis; for example, during regeneration after tissue injury. Examples of intestinal cells with ISC potential are label-retaining Paneth cell precursors and Delta-like 1-positive (DLL1 + ) secretory precursors. The functional ISC compartment is the number of cells with ISC activity corrected for their relative contribution to the total output of the stem cell compartment. Mutations that are commonly found in colorectal cancer (CRC), such as adenomatous polyposis coli ( APC ) inactivation and KRAS activation, act on ISC dynamics and give a competitive advantage to the cell in which they occur. The benefit of mutated ISCs over wild-type ISCs is not absolute, and mutated ISCs are frequently outcompeted by wild-type ISCs. CRCs contain cells with stem cell-like activity; however, the frequency of these cells remains unknown, as does the importance of these cells for the biology of CRCs. Differentiated cancer cells and cancer stem cells are in constant flux, which is influenced by signals that emanate from the tumour stroma. This Review discusses recent studies that offer quantitative insights into the dynamics of intestinal stem cell behaviour that govern homeostasis. These studies provide the necessary baseline parameters such that we can begin to understand stem cell behaviour during colorectal cancer development. Intestinal stem cells (ISCs) and colorectal cancer (CRC) biology are tightly linked in many aspects. It is generally thought that ISCs are the cells of origin for a large proportion of CRCs and crucial ISC-associated signalling pathways are often affected in CRCs. Moreover, CRCs are thought to retain a cellular hierarchy that is reminiscent of the intestinal epithelium. Recent studies offer quantitative insights into the dynamics of ISC behaviour that govern homeostasis and thereby provide the necessary baseline parameters to begin to apply these analyses during the various stages of tumour development.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids’ phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose–glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.This protocol for clearing and high-resolution 3D imaging of entire organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC. Ponsioen et al. use a FRET‐based ERK biosensor EKAREN5 in patient‐derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways.

The glycolytic activity of Paneth cells provides lactate, which is required by self-renewing intestinal stem cells for oxidative metabolism to activate p38 MAP kinase, ensuring regeneration of a mature crypt. Metabolism and gut regeneration Small intestine crypts are regenerated throughout life thanks to self-renewing stem cells located at the bottom of crypts. Differentiated Paneth cells provide the signalling molecules that modulate the regenerative properties of these stem cells. The influence of metabolism on the self-renewal of the crypt has not been studied in great detail. Burgering and colleagues now show that, whereas intestinal stem cells rely on mitochondrial activity for their metabolic needs, Paneth cells use glycolysis, a process that provides the lactate that is required by the stem cells for their oxidative metabolism. This activates the p38 MAP kinase to ensure regeneration of a mature crypt. The findings suggest that the metabolism of certain intestinal cells has an important role in supporting stem cell function. The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5 + crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt 1 . Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5 + CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5 + CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5 + CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5 + CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5 + CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

The maintenance of stem‐cell‐driven tissue homeostasis requires a balance between the generation and loss of cell mass. Adult stem cells have a close relationship with the surrounding tissue—known as their niche—and thus, stem‐cell studies should preferably be performed in a physiological context, rather than outside their natural environment. The mouse is an attractive model in which to study adult mammalian stem cells, as numerous experimental systems and genetic tools are available. In this review, we describe strategies commonly used to identify and functionally characterize adult stem cells in mice and discuss their potential, limitations and interpretations, as well as how they have informed our understanding of adult stem‐cell biology. An accurate interpretation of physiologically relevant stem‐cell assays is crucial to identify adult stem cells and elucidate how they self‐renew and give rise to differentiated progeny. Physiologically relevant assays are crucial to identify adult stem cells and understand how they self‐renew and give rise to differentiated progeny. The most commonly used strategies are discussed here, as well as how they have contributed to our understanding of stem‐cell biology.

The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By \"lineage retracing\" using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat—containing heterotrimeric guanine nucleotide—binding protein—coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5 + cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5 + cells as well as all other adenoma cell types. The Lgr5 + cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.

Paneth cells carve out a niche Paneth cells, specialized cells found in the intestinal epithelium, are known to protect stem cells by producing bactericidal compounds. Now another crucial function is reported: they provide the essential niche signals (EGF/TGFα, Notch and Wnt) for Lgr5 -expressing stem cells in the small intestine. Multipotent stem cells expressing Lgr5 generate all intestinal epithelium cell types — Paneth cells included. Stem-cell niches are often seen as pre-existing sites to which stem cells migrate; this work shows that intestinal stem cells receive niche support from their own progeny. Multipotent stem cells expressing Lgr5 are known to generate all cell types of the intestinal epithelium (enterocytes, goblet cells, Paneth cells and enteroendocrine cells). A new study shows that Paneth cells have an essential role for intestinal crypt and stem cell maintenance by supplying essential niche signals to the Lgr5-expressing cells. Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms 1 , 2 . Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins 3 . Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt–villus organoids in the absence of non-epithelial niche cells 4 . Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro . CD24 + Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24 + cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types. Intratumour heterogeneity (ITH) is associated with worse prognosis in cancer, and efficient frameworks to measure it are needed. Here the authors develop a method to estimate copy number heterogeneity, and propose that it is driven by chromosomal instability and can predict pan-cancer survival.

The authors use in vivo imaging to examine astrocyte dynamics after a cortical injury over the course of several weeks. They reveal a heterogeneity in astrocyte responses and show that astrocytes do not migrate toward the injury site, but instead proliferate in the juxtavascular region. Astrocytes are thought to have important roles after brain injury, but their behavior has largely been inferred from postmortem analysis. To examine the mechanisms that recruit astrocytes to sites of injury, we used in vivo two-photon laser-scanning microscopy to follow the response of GFP-labeled astrocytes in the adult mouse cerebral cortex over several weeks after acute injury. Live imaging revealed a marked heterogeneity in the reaction of individual astrocytes, with one subset retaining their initial morphology, another directing their processes toward the lesion, and a distinct subset located at juxtavascular sites proliferating. Although no astrocytes actively migrated toward the injury site, selective proliferation of juxtavascular astrocytes was observed after the introduction of a lesion and was still the case, even though the extent was reduced, after astrocyte-specific deletion of the RhoGTPase Cdc42. Thus, astrocyte recruitment after injury relies solely on proliferation in a specific niche.