Activating mutations in the gene encoding β-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of β-catenin (β-catnc) and over-activation of the Wnt/β-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the β-catnc cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these β-catnc cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the β-catnc cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-0957-9) contains supplementary material, which is available to authorized users.

Cyclin‐dependent kinase (Cdk)7, the catalytic subunit of the Cdk‐activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)‐mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7‐expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age‐related phenotypes, including telomere shortening and early death.

TRF1 is a component of the shelterin complex that protects chromosome ends. TRF1 deficiency leads to early embryonic lethality and to severe organ atrophy when deleted in adult tissues. Here we generate a reporter mouse carrying a knock-in eGFP-TRF1 fusion allele to study the role of TRF1 in stem cell biology and tissue homeostasis. We find that eGFP-TRF1 expression in mice is maximal in known adult stem cell compartments and show that TRF1 ensures their functionality. eGFP-TRF1 is highly expressed in induced pluripotent stem cells, uncoupled from the telomere elongation associated with reprogramming. Selection of eGFP-TRF1-high induced pluripotent stem cells correlates with higher pluripotency as indicated by their ability to form teratomas and chimeras. We further show that TRF1 is necessary for both induction and maintenance of pluripotency, and that TRF1 is a direct transcriptional target of Oct3/4.

Murine embryonic stem (ES) cells have unusually long telomeres, much longer than those in embryonic tissues. Here we address whether hyper-long telomeres are a natural property of pluripotent stem cells, such as those present at the blastocyst inner cell mass (ICM), or whether it is a characteristic acquired by the in vitro expansion of ES cells. We find that ICM cells undergo telomere elongation during the in vitro derivation of ES-cell lines. In vivo analysis shows that the hyper-long telomeres of morula-injected ES cells remain hyper-long at the blastocyst stage and longer than telomeres of the blastocyst ICM. Telomere lengthening during derivation of ES-cell lines is concomitant with a decrease in heterochromatic marks at telomeres. We also found increased levels of the telomere repeat binding factor 1 (TRF1) telomere-capping protein in cultured ICM cells before telomere elongation occurs, coinciding with expression of pluripotency markers. These results suggest that high TRF1 levels are present in pluripotent cells, most likely to ensure proficient capping of the newly synthesized telomeres. These results highlight a previously unnoticed difference between ICM cells at the blastocyst and ES cells, and suggest that abnormally long telomeres in ES cells are likely to result from continuous telomere lengthening of proliferating ICM cells locked at an epigenetic state associated to pluripotency.ouse embryonic stem (ES) cells are pluripotent, proliferate indefinitely, and bear very long telomeres (1-3). ES cells emerge from preimplantation blastocyst-stage embryos (4), but how this process takes place is largely unknown. In previous studies, we observed that telomeres of mouse ES cells were much longer than those of mouse embryonic fibroblasts (MEFs) of the same genetic background (5), which are typically obtained at embryonic day 13.5 (E13.5). This observation raised the issue of whether blastocyst inner cell mass (ICM) cells, which are the natural equivalents of ES cells, also have hyper-long telomeres. If this is the case, then telomeres must shorten during fetal development, despite high telomerase activity (6-8). An alternative explanation emerges, however, that hyper-long telomeres in ES cell are aberrant and may result from the in vitro establishment and expansion of ES cells.ES-like pluripotent stem cells can be generated from differentiated cells (i.e., MEFs) by using defined factors, giving rise the so-called induced pluripotent stem (iPS) cells, which are considered functional equivalents of ES cells (9-16). We recently showed that iPS telomeres increase in length during and after nuclear reprogramming until reaching ES cell hyper-long telomeres. This elongation process occurs concomitantly to lower density of trimethylated histones H3K9 and H4K20 at the telomeric chromatin (5). Furthermore, hyper-long telomeres were not observed in iPS cells derived from first-generation telomerasedeficient MEFs, indicating that they do not originate from a selective reprogramming of a subset of parental cells with v...

SummarySox4 expression is restricted in mammals to embryonic structures and some adult tissues, such as lymphoid organs, pancreas, intestine, and skin. During embryogenesis, Sox4 regulates mesenchymal and neural progenitor survival, as well as lymphocyte and myeloid differentiation, and contributes to pancreas, bone, and heart development. Aberrant Sox4 expression is linked to malignant transformation and metastasis in several types of cancer. To understand the role of Sox4 in the adult organism, we first generated mice with reduced whole-body Sox4 expression. These mice display accelerated aging and reduced cancer incidence. To specifically address a role for Sox4 in adult stem cells, we conditionally deleted Sox4 (Sox4cKO) in stratified epithelia. Sox4cKO mice show increased skin stem cell quiescence and resistance to chemical carcinogenesis concomitantly with downregulation of cell cycle, DNA repair, and activated hair follicle stem cell pathways. Altogether, these findings highlight the importance of Sox4 in regulating adult tissue homeostasis and cancer.

SummaryThe shelterin complex protects telomeres by preventing them from being degraded and recognized as double‐strand DNA breaks. TRF1 is an essential component of shelterin, with important roles in telomere protection and telomere replication. We previously showed that TRF1 deficiency in the context of different mouse tissues leads to loss of tissue homeostasis owing to impaired stem cell function. Here, we show that TRF1 levels decrease during organismal aging both in mice and in humans. We further show that increasing TRF1 expression in both adult (1‐year‐old) and old (2‐year‐old) mice using gene therapy can delay age‐associated pathologies. To this end, we used the nonintegrative adeno‐associated serotype 9 vector (AAV9), which transduces the majority of mouse tissues allowing for moderate and transient TRF1 overexpression. AAV9‐TRF1 gene therapy significantly prevented age‐related decline in neuromuscular function, glucose tolerance, cognitive function, maintenance of subcutaneous fat, and chronic anemia. Interestingly, although AAV9‐TRF1 treatment did not significantly affect median telomere length, we found a lower abundance of short telomeres and of telomere‐associated DNA damage in some tissues. Together, these findings suggest that rescuing naturally decreased TRF1 levels during mouse aging using AAV9‐TRF1 gene therapy results in an improved mouse health span.

Induced pluripotent stem cells (iPSCs) can be differentiated in vitro and in vivo to all cardiovascular lineages and are therefore a promising cell source for cardiac regenerative therapy. However, iPSC lines do not all differentiate into cardiomyocytes (CMs) with the same efficiency. Here, we show that telomerase-competent iPSCs with relatively long telomeres and high expression of the shelterin-complex protein TRF1 (iPSC ) differentiate sooner and more efficiently into CMs than those with relatively short telomeres and low TRF1 expression (iPSC ). Ascorbic acid, an enhancer of cardiomyocyte differentiation, further increases the cardiomyocyte yield from iPSC but does not rescue the cardiomyogenic potential of iPSC . Interestingly, although iPSCs differentiate very poorly to the mesoderm and endoderm lineages, they differentiate very efficiently to the ectoderm lineage, indicating that cell fate can be determined by in vitro selection of iPSCs with different telomere content. Our findings highlight the importance of selecting iPSCs with ample telomere reserves in order to generate high numbers of CMs in a fast, reliable, and efficient way. Stem Cells 2017;35:362-373.

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