A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.Epidermal stem cells ensure that skin homeostasis is maintained. Murine epidermal stem cells are located either at the permanent portion of the hair follicle-termed the bulge-and are exclusively responsible for hair cycling [1][2][3][4] ; or at the junction between the epidermis and the hair follicle (isthmus), and feed into the epidermis and sebaceous glands [5][6][7] . In addition, a continuous proliferation of basal interfollicular epidermal cells ensures daily epidermal maintenance 8 .Bulge stem cells undergo bouts of activation followed by periods of dormancy, to establish hair follicle cycling. Robust TGF-b and Bmp signals act as 'activation breaks', rendering bulge cells dormant during the resting phase of the hair cycle (telogen) [9][10][11] . At the onset of the growth phase (anagen), bulge cells respond to Wnt signals by migrating into the lower proliferative hair germ region, where they contribute to follicle growth [12][13][14][15] . Subsequently, at mid-anagen, the bulge undergoes a second round of activation, which replenishes cells lost at the onset of anagen 2,3 . However, the response of bulge stem cells to activating stimuli is a heterogeneous process, as only a subset of them become active during either stage of activation 12,13 . The nature of such niche heterogeneity is currently unknown. Importantly, perturbing the equilibrium between the responsive and non-responsive stem cell states causes tissue malfunction and increases the risk of carcinogenesis [16][17][18][19][20] .Here, we analysed the role of the molecular clock in fine-tuning the function of epidermal stem cells. The mammalian clock machinery anticipates and synchronizes vital functions related to the physiological circadian needs of the organism 21 . The core molecular clock is established by a positive limb, composed of heterodimers of the transcription fa...

In general, plasma membrane integral proteins, such as the membrane-anchored growth factor proTGF-alpha, are assumed to be transported to the cell surface via a nonregulated, constitutive pathway. proTGF-alpha C-terminal mutants are retained in an early secretory compartment. Here, using a two-hybrid screen, we identify two TACIPs (proTGF-alpha cytoplasmic domain-interacting proteins) that contain PDZ domains and do not interact with proTGF-alpha C-terminal mutants. The binding specificity of one of them, TACIP18 (previously identified and named Syntenin or mda-9), coincides with that of the component that possibly mediates the normal trafficking of proTGF-alpha. TACIP18 colocalizes and interacts specifically with immature, intracellular forms of proTGF-alpha. Therefore, it appears that the interaction of TACIP18 with proTGF-alpha in the early secretory pathway is necessary for the targeting of the latter to the cell surface.

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus. Analysis of LGR5‐EGFP + cells isolated from organoid‐derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage‐tracing experiments showed that LGR5+ CRC cells self‐renew and generate progeny over long time periods that undergo differentiation toward mucosecreting‐ and absorptive‐like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.