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Skeletal progenitor/stem cells (SSCs) play a critical role in postnatal bone growth and maintenance. Telomerase (Tert) activity prevents cellular senescence and is required for maintenance of stem cells in self‐renewing tissues. Here we investigated the role of mTert‐expressing cells in postnatal mouse long bone and found that mTert expression is enriched at the time of adolescent bone growth. mTert‐GFP+ cells were identified in regions known to house SSCs, including the metaphyseal stroma, growth plate, and the bone marrow. We also show that mTert‐expressing cells are a distinct SSC population with enriched colony‐forming capacity and contribute to multiple mesenchymal lineages, in vitro. In contrast, in vivo lineage‐tracing studies identified mTert + cells as osteochondral progenitors and contribute to the bone‐forming cell pool during endochondral bone growth with a subset persisting into adulthood. Taken together, our results show that mTert expression is temporally regulated and marks SSCs during a discrete phase of transitional growth between rapid bone growth and maintenance., In long bones, osteochondral progenitor cells function during rapid bone growth while osteoadipogenic progenitors act during bone maintenance. We showed that mTert is expressed in an age‐dependent manner and marks growth‐associated osteochondral progenitor cells during transitional growth, a discrete time period between rapid bone growth and bone maintenance.

Sufficient nutrition is critical for reproduction. We have previously shown that leptin, a circulating indicator of fat stores, signals to pituitary gonadotropes to maintain gonadotropin releasing hormone receptor (GnRHR) protein levels in female mice. We hypothesized that this process is post-transcriptional, happening primarily through regulation of the RNA-binding protein Musashi (MSI). We showed that MSI binds to Gnrhr and inhibits translation, and a gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi1/2-null) leads to increased GnRHR protein levels. This culminates in dysregulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH). We have recently identified other gonadotrope and pituitary targets of MSI. We therefore suspected that MSI plays a role in both the maturation of gonadotropes and the normal cyclic regulation of gonadotropes. We hypothesized that the deletion of MSI would lead to downstream effects on (1) the composition of the gonadotrope population and (2) the molecular landscape of these cells. Using our adult, diestrous Gon-Msi1/2-null females, we performed single-cell RNA-sequencing on methanol-fixed dispersed pituitary cells. Libraries were made from two control pools and two mutant pools (n=3 pituitaries/pool) using 10x Genomics v3.1 Single-Cell Gene Expression technology and initially sequenced on an Illumina Next-seq mid-output flow-cell, yielding 5,000 reads/cell. Subsequent high-output sequencing obtained 25,000 reads/cell. We recovered single-cell mRNA transcript information from 18,206 control pituitary cells and 16,255 Gon-Msi1/2-null cells. Our analyses revealed that the Gon-Msi1/2-null pools had a higher % of cells expressing Fshb, as well as an expected significant drop in Msi2-expressing gonadotropes and no change in Lhb-expressing cells. We have recently identified Fshb as an MSI target in silico, and qRT-PCR of female pituitary lysate immunoprecipitated with anti-MSI1 shows a 7-fold enrichment in Fshb mRNA. We identified differentially expressed genes comparing the control and Gon-Msi1/2-null gonadotrope clusters. Using Gene Ontology analyses, the Gon-Msi1/2-null gonadotrope cluster appears to have aberrant expression of mRNAs involved in protein folding and cellular responses to nutrients. Our high-output sequencing has allowed us to achieve 25,000 reads/cell and will provide greater resolution of the role of Musashi in control of gonadotrope function. Taken together, our data indicate that Musashi influences the molecular landscape and subsequent physiology of the female gonadotrope. We have identified potential gonadotrope-specific MSI targets, including pathways that may underlie the dysregulated gonadotropin production and secretion seen in our Gon-Msi1/2-null females. Future studies will compare pubertal and adult females, as well as females from different estrous cycle stages.

Efimova et al. show that adenomatous polyposis coli (APC) at microtubule tips triggers assembly of a branched actin network when the microtubule hits the plasma membrane in neuronal growth cones. These findings uncover a new mechanism of microtubule-dependent cell navigation., Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.

Summary Two principal stem cell pools orchestrate the rapid cell turnover in the intestinal epithelium. Rapidly cycling Lgr5+ stem cells are intercalated between the Paneth cells at the crypt base (CBCs) and injury-resistant reserve stem cells reside above the crypt base. The intermediate filament Keratin 15 (Krt15) marks either stem cells or long-lived progenitor cells that contribute to tissue repair in the hair follicle or the esophageal epithelium. Herein, we demonstrate that Krt15 labels long-lived and multipotent cells in the small intestinal crypt by lineage tracing. Krt15+ crypt cells display self-renewal potential in vivo and in 3D organoid cultures. Krt15+ crypt cells are resistant to high-dose radiation and contribute to epithelial regeneration following injury. Notably, loss of the tumor suppressor Apc in Krt15+ cells leads to adenoma and adenocarcinoma formation. These results indicate that Krt15 marks long-lived, multipotent, and injury-resistant crypt cells that may function as a cell of origin in intestinal cancer., Highlights • Krt15 marks multipotent and self-renewing crypt cells in the mouse small intestine • Krt15+ crypt cells are radio-resistant and contribute to regeneration following injury • Apc loss in Krt15+ cells leads to intestinal adenoma and adenocarcinoma formation • Krt15+ cells may function as a cell of origin in intestinal cancer, In this article, Rustgi and colleagues identify a new population of long-lived cells within the intestinal crypt, marked by the Krt15 promoter. Krt15+ cells are self-renewing, multipotent, and radio-resistant cells that facilitate recovery from radiation-induced injury. Furthermore, such cells, when targeted with Apc loss, undergo transformation to adenomas and adenocarcinomas.

Summary The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

Somatic stem cells maintain tissue homeostasis by organizing themselves in such a way that they can maintain proliferative output while simultaneously protecting themselves from DNA damage that may lead to oncogenic transformation. There is considerable debate as to how such stem cell compartments are organized. Burgeoning evidence from the small intestine and colon provides support for a two-stem cell model involving actively proliferating, but injury-sensitive stem cell and a rare, injury-resistant pool of quiescent stem cells. Parallel to this evidence, recent studies have revealed considerable plasticity within the intestinal stem cell compartment. We discuss the evidence for plasticity and hierarchy within the intestinal stem cell compartment and how these properties govern tissue regeneration and contribute to oncogenic transformation leading to colorectal cancers.

The recent generation of induced neurons by direct lineage conversion holds promise for in vitro modelling of sporadic Alzheimer's disease. Here, we report the generation of induced neuron-based model of sporadic Alzheimer's disease in mice and humans, and used this system to explore the pathogenic mechanisms resulting from the sporadic Alzheimer's disease risk factor apolipoprotein E (APOE) ɛ3/4 allele. We show that mouse and human induced neurons overexpressing mutant amyloid precursor protein in the background of APOE ɛ3/4 allele exhibit altered amyloid precursor protein (APP) processing, abnormally increased production of amyloid-β42 and hyperphosphorylation of tau. Importantly, we demonstrate that APOE ɛ3/4 patient induced neuron culture models can faithfully recapitulate molecular signatures seen in APOE ɛ3/4-associated sporadic Alzheimer's disease patients. Moreover, analysis of the gene network derived from APOE ɛ3/4 patient induced neurons reveals a strong interaction between APOE ɛ3/4 and another Alzheimer's disease risk factor, desmoglein 2 (DSG2). Knockdown of DSG2 in APOE ɛ3/4 induced neurons effectively rescued defective APP processing, demonstrating the functional importance of this interaction. These data provide a direct connection between APOE ɛ3/4 and another Alzheimer's disease susceptibility gene and demonstrate in proof of principle the utility of induced neuron-based modelling of Alzheimer's disease for therapeutic discovery.

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia.

Critical telomere shortening (for example, secondary to partial telomerase deficiency in the rare disease dyskeratosis congenita) causes tissue pathology, but underlying mechanisms are not fully understood. Mice lacking telomerase (for example, mTR−/− telomerase RNA template mutants) provide a model for investigating pathogenesis. In such mice, after several generations of telomerase deficiency telomeres shorten to the point of uncapping, causing defects most pronounced in high-turnover tissues including intestinal epithelium. Here we show that late-generation mTR−/− mutants experience marked downregulation of Wnt pathway genes in intestinal crypt epithelia, including crypt base columnar stem cells and Paneth cells, and in underlying stroma. The importance of these changes was revealed by rescue of crypt apoptosis and Wnt pathway gene expression upon treatment with Wnt pathway agonists. Rescue was associated with reduced telomere-dysfunction-induced foci and anaphase bridges, indicating improved telomere capping. Thus a mutually reinforcing feedback loop exists between telomere capping and Wnt signalling, and telomere capping can be impacted by extracellular cues in a fashion independent of telomerase., Mice lacking telomerase provide a model to study pathogenesis caused by critical telomere shortening. Here, the authors provide evidence that telomere shortening causes downregulation of Wnt signalling in intestinal crypts and that defects can be partially rescued by treatment with Wnt agonists.

Current studies offer little insight on how epigenetic remodeling of bone-specific chromatin maintains bone mass in vivo. Understanding this gap and precise mechanism is pivotal for future therapeutic innovation to prevent bone loss. Recently, we found that low bone mass is associated with decreased H3K27 acetylation (activating histone modification) of bone specific gene promoters. Here, we aim to elucidate the epigenetic mechanisms by which a miRNA cluster controls bone synthesis and homeostasis by regulating chromatin accessibility and H3K27 acetylation. In order to decipher the epigenetic axis that regulates osteogenesis, we studied a drug inducible anti-miR-23a cluster (miR-23a ClZIP) knockdown mouse model. MiR-23a cluster knockdown (heterozygous) mice developed high bone mass. These mice displayed increased expression of Runx2 and Baf45a, essential factors for skeletogenesis; and decreased expression of Ezh2, a chromatin repressor indispensable for skeletogenesis. ChIP assays using miR-23a Cl knockdown calvarial cells revealed a BAF45A-EZH2 epigenetic antagonistic mechanism that maintains bone formation. Together, our findings support that the miR-23a Cl connection with tissue-specific RUNX2-BAF45A-EZH2 function is a novel molecular epigenetic axis through which a miRNA cluster orchestrates chromatin modification to elicit major effects on osteogenesis in vivo.

The +4 cells in intestinal crypts are DNA damage-resistant and contribute to regeneration. However, their exact identity and the mechanism underlying +4 cell-mediated regeneration remain unclear. Using lineage tracing, we show that cells marked by anMsi1reporter (Msi1+) are enriched at the +4 position in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that theMsi1+cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation ofMsi1+cells is characterized by low-to-negativeLgr5expression and is more rapidly cycling thanLgr5highradio-sensitive crypt base columnar stem cells (CBCs); they enable fast repopulation of the intestinal epithelium independent of CBCs that are largely depleted after irradiation. Furthermore, relative to CBCs,Msi1+cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistantMsi1+cells are rapidly cycling ISCs that maintain and regenerate the intestinal epithelium.

Leptin is a critical mediator of metabolic regulation of the hypothalamic-pituitary gonadal (HPG) axis. We have previously shown that leptin is responsible for the optimal expression of GnRHR, a rate-limiting component of the reproductive process. Female mice lacking leptin receptors (Lepr-null) specifically on gonadotropes are sub-fertile. Their reduced GnRHR proteins and normal Gnrhr mRNA levels suggest leptin’s actions on gonadotropes are post-transcriptional. A clue about a candidate translational regulator is seen in our studies showing that Lepr-null gonadotropes have increased expression of Musashi (MSI), which has binding sites within the Gnrhr 3’ UTR. Furthermore, leptin reduced MSI expression specifically in gonadotropes and increased GnRHR expression. We hypothesized that MSI may repress Gnrhr mRNA translation and that leptin alleviates this repression. To determine the effects of MSI on the HPG axis, we developed a gonadotrope-Msi1/2-null mouse line and compared mutant females (MUT) to their littermate controls (CTL) on the morning of diestrus, when the pituitary GnRHR protein levels should reach a peak. The levels of GnRHR proteins (measured by EIA) are significantly increased (1.6X) in the pituitary of the mutant females (CTL: 1.355 ± 0.11 ng/ml vs MUT: 2.202 ± 0.16 ng/ml, p=.0006), with no change in mRNA. The gonadotrope-Msi1/2-null females are subfertile, with litter sizes of 3 ± 0.4 pups, with the first litter at around day 40 and an average of 41 day delay between litters. To understand the downstream effect of the MSI knockout on gonadotropin levels, we measured pituitary and serum LH and FSH protein levels (Luminex EIA) and mRNA (qPCR). Serum FSH levels are decreased by >50% in mutant females (CTL: 0.947 ± 0.14 ng/mL vs MUT: 0.406 ± 0.05 ng/ml, p=.0049), but FSH stores and Fsh mRNAs are unchanged. Additionally, we observed a 2.2X increase in the pituitary LH protein content in mutant females (CTL: 0.328 ± 0.11 ng/ml vs MUT: 0.729 ± 0.09 ng/ml, p=.0174), but no changes in serum LH or Lh mRNA levels. These studies thus show that, as a repressor of GnRHR translation, Musashi can also regulate expression of gonadotropins. In the gonadotrope Lepr-null model, we hypothesized that leptin signals were needed to de-repress Musashi actions and allow GnRHR translation. In contrast, the gonadotrope-MSI-null mice over-express GnRHR, which confirms this role for MSI as a GnRHR regulator. We propose that the increased expression of GnRHR in the gonadotrope MSI-null animals causes the increased LH content, and disrupted FSH secretion, resulting in a higher serum LH:FSH ratio and subfertility. Collectively these data suggest that MSI regulation is necessary for optimal fertility in the adult female mouse. Future studies will determine the impact of loss of MSI on gonadotrope function throughout the estrous cycle.

B cell receptor engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that each cell navigate through checkpoints that control its survival, cell cycle entry, and proliferative expansion. Dynamic changes in BCR-induced calcium concentration critically regulate these checkpoints. Here we establish that variations in BCR signaling strength are encoded as calcium signals that tune each cell’s fate by dynamically regulating NF-κB, NFAT, and mTORC1 dependent control of gene expression. We find that weak BCR signaling induces apoptosis by failing to activate c-Rel-dependent Bcl-xL expression. Progressively stronger signals, which generate quantitatively distinct calcium signals, are required to promote c-Rel dependent survival, drive metabolic reprogramming, initiate cell cycle entry, and drive proliferation via NFAT-, mTORC1-, and Myc-dependent mechanisms. Finally, we establish the mechanism by which CD40 and PAMP costimulation decrease the calcium threshold for these key steps of B cell activation and differentiation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, our results have important implications for understanding the pathogenesis of aberrant B cell differentiation.

Cerebral cavernous malformation (CCM) is a genetic, cerebrovascular disease. Familial CCM is caused by genetic mutations in KRIT1, CCM2 or PDCD10. Notably, disease onset is earlier and more severe in individuals with PDCD10 mutations. Recent studies have shown that lesions arise from excess Mitogen-Activated Protein Kinase Kinase Kinase 3 (MEKK3) signaling downstream of Toll-like Receptor 4 (TLR4) stimulation by lipopolysaccharide (LPS) derived from the gut microbiome. These findings suggest a gut-brain CCM disease axis but fail to define it or explain the poor prognosis of patients with PDCD10 mutations. Here, we demonstrate that the gut barrier is a primary determinant of CCM disease course, independent of microbiome configuration, that explains the increased severity of CCM disease associated with PDCD10 deficiency. Chemical disruption of the gut barrier with dextran sodium sulfate augments CCM formation in a mouse model, as does genetic loss of Pdcd10, but not Krit1, in gut epithelial cells. Loss of gut epithelial Pdcd10 results in disruption of the colonic mucosal barrier. Accordingly, loss of Mucin-2 or exposure to dietary emulsifiers that reduce the mucus barrier increase CCM burden analogous to loss of Pdcd10 in gut epithelium. Finally, we show that treatment with dexamethasone potently inhibits CCM formation in mice due to the combined effect of action at both brain endothelial cells and gut epithelial cells. These studies define a gut-brain disease axis in an experimental model of CCM in which a single gene is required for two critical components: gut epithelial function and brain endothelial signaling.

The cyclic expression of gonadotropin releasing-hormone receptors (GnRHR), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary gonadotropes is critical in the female reproductive process. We have shown that the translational regulator Musashi (MSI) binds to Gnrhr mRNA and inhibits its translation, and the gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi-null) leads to increased pituitary GnRHR protein levels. An in silico analysis of gonadotropin mRNAs revealed 5 different MSI binding elements in the 3’UTR of Fshb mRNA. We hypothesize that, in addition to Gnrhr, MSI may also bind and repress Fshb mRNA translation in the gonadotropes. To test if MSI does target the Fshb transcript in the pituitary, we performed RNA immunoprecipitation (IP) on pooled control female mouse pituitaries using a MSI1 antibody and measured Fshb mRNA by qRT-PCR. To study the in vivo effects of MSI on Fshb, we harvested the pituitaries of the Gon-Msi-null (MUT) female mice and their littermate controls (CTL) during the estrous cycle. We collected serum and protein for EIAs to measure the levels of FSH and LH, and RNA for Fshb qRT-PCR. We harvested proestrous ovaries and fixed them for embedding, sectioning, and H&E staining. Our RNA IP experiments show a 7-fold enrichment for Fshb with the MSI1 antibody. The Gon-Msi-null females have significantly higher pituitary FSH protein content than controls on estrous morning (MUT: 4.8±1.3 vs. CTL: 1.8±2.6 ng/ml/μg protein, p

Active DNA demethylation via Ten-eleven Translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically-engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

Mammary epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of contractile myoepithelial cells with mesenchymal properties. These two compartments interact throughout mammary morphogenesis to form branching ducts during puberty and terminate in secretory alveoli during lactation. It is not known how the myoepithelial cell lineage is specified, nor how signals in myoepithelial cells contribute to lactogenesis. Here, we show that Numb and Numbl are enriched in mammary myoepithelial cells, with their expression peaking during pregnancy. We use conditional Numb- and Numbl-knockout mouse models to demonstrate that loss of Numb/Numbl compromised the myoepithelial layer and expanded the luminal layer, led epithelial cells to undergo epithelial-to-mesenchymal transition, and resulted in lactation failure as a result of abnormal alveolar formation during pregnancy. Numb and Numbl function via repression of the Notch signaling pathway and of the p53-p21 axis during mammary gland development. These findings highlight the importance of Numb and Numbl in the control of myoepithelial cell fate determination, epithelial identity, and lactogenesis.-Zhang Y., Li, F., Song, Y., Sheng, X., Ren, F., Xiong, K., Chen, L., Zhang, H., Liu, D., Lengner, C. J., Xue, L., Yu, Z. Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis.

Background Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. Results Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base. Conclusions Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease., Several studies have recently demonstrated the role of the MSI2 RNA binding protein in normal and malignant haematopoietc stem cells. In this study, the authors show that MSI2 is required for maintaining myelodysplastic syndrome stem cells in mice and that MSI2 expression predicts poor prognosis in patients affected by this disease.

This work demonstrates that Msi RNA-binding proteins are required for the exit of reserve intestinal stem cells from quiescence and further illustrates the importance of maintaining a quiescent pool of stem cells for regeneration of the intestinal epithelium after injury., Regeneration of the intestinal epithelium is driven by multiple intestinal stem cell (ISC) types, including an active, radiosensitive Wnthigh ISC that fuels turnover during homeostasis and a reserve, radioresistant Wntlow/off ISC capable of generating active Wnthigh ISCs. We examined the role of the Msi family of oncoproteins in the ISC compartment. We demonstrated that Msi proteins are dispensable for normal homeostasis and self-renewal of the active ISC, despite their being highly expressed in these cells. In contrast, Msi proteins are required specifically for activation of reserve ISCs, where Msi activity is both necessary and sufficient to drive exit from quiescence and entry into the cell cycle. Ablation of Msi activity in reserve ISCs rendered the epithelium unable to regenerate in response to injury that ablates the active stem cell compartment. These findings delineate a molecular mechanism governing reserve ISC quiescence and demonstrate a necessity for the activity of this rare stem cell population in intestinal regeneration.

SummaryMembers of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

Recent work generating induced dopaminergic (iDA) neurons using direct lineage reprogramming potentially provides a novel platform for the study and treatment Parkinson's disease (PD). However, one of the most important issues for iDA-based applications is the degree to which iDA neurons resemble the molecular and functional properties of their endogenous DA neuron counterparts. Here we report that the homogeneity of the reprogramming gene expression system is critical for the generation of iDA neuron cultures that are highly similar to endogenous DA neurons. We employed an inducible system that carries iDA-inducing factors as defined transgenes for direct lineage reprogramming to iDA neurons. This system circumvents the need for viral transduction, enabling a more efficient and reproducible reprogramming process for the generation of genetically homogenous iDA neurons. We showed that this inducible system generates iDA neurons with high similarity to their bona fide in vivo counterparts in comparison to direct infection methods. Thus, our results suggest that homogenous expression of exogenous genes in direct lineage reprogramming is critical for the generation of high quality iDA neuron cultures, making such culture systems a valuable resource for iDA-based drug screening and, ultimately, potential therapeutic intervention in PD.

SUMMARY B cell receptor (BCR) engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that a cell survive, enter the cell cycle, and proliferate. We establish that quantitatively distinct Ca2+ signals triggered by variations in the extent of BCR engagement dynamically regulate these transitions by controlling nuclear factor κB (NF-κB), NFAT, and mTORC1 activity. Weak BCR engagement induces apoptosis by failing to activate NF-κB-driven anti-apoptotic gene expression. Stronger signals that trigger more robust Ca2+ signals promote NF-κB-dependent survival and NFAT-, mTORC1-, and c-Myc-dependent cell-cycle entry and proliferation. Finally, we establish that CD40 or TLR9 costimulation circumvents these Ca2+-regulated checkpoints of B cell activation and proliferation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, these results have important implications for understanding the pathogenesis of aberrant B cell activation and differentiation and therapeutic approaches to target these responses., Graphical Abstract, In Brief Berry et al. establish that variations in the strength of BCR engagement are encoded as quantitatively distinct calcium signals that tune B cell fates by dynamically regulating NF-κB, NFAT, and mTORC1 activity. Targeting calcium signaling may thereby serve as an effective treatment strategy for regulating normal and pathological B cell activation.

NAD+ is essential for redox reactions in energy metabolism and necessary for DNA repair and epigenetic modification. Humans require sufficient amounts of dietary niacin (nicotinic acid, nicotinamide, and nicotinamide riboside) for adequate NAD+ synthesis. In contrast, mice easily generate sufficient NAD+ solely from tryptophan through the kynurenine pathway. We show that transgenic mice with inducible expression of human alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) become niacin dependent similar to humans when ACMSD expression is high. On niacin-free diets, these acquired niacin dependency (ANDY) mice developed reversible, mild-to-severe NAD+ deficiency, depending on the nutrient composition of the diet. NAD deficiency in mice contributed to behavioral and health changes that are reminiscent of human niacin deficiency. This study shows that ACMSD is a key regulator of mammalian dietary niacin requirements and NAD+ metabolism and that the ANDY mouse represents a versatile platform for investigating pathologies linked to low NAD+ levels in aging and neurodegenerative diseases.

MicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3′-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a Cl(WT/ZIP)) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stage-specific HoxA factor expression during osteogenesis.

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B. In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B–IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.

Summary Expression of type I interferons (IFNs) can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

Intestinal organoids offer great promise for modeling intestinal diseases; however, harvesting intestinal tissue is invasive and directed hPSC differentiation protocols are laborious and costly. In this issue of Cell Stem Cell, Miura and Suzuki (2017) describe the direct conversion of somatic cells from both mice and humans into robust intestinal epithelial tissue.

Summary Aging is a complex process associated with a decline in functionality of adult stem cells affecting tissue homeostasis and regeneration. Calorie restriction (CR) is the only experimental manipulation known to extend lifespan and reduce the incidence of age-related disorders across numerous species. These benefits are likely mediated, at least in part, through the preservation of stem cell function. Here, we show that CR enhances the regenerative capacity of the intestinal epithelium through preservation of an injury-resistant reserve intestinal stem cell (ISC) pool. Cell-autonomous activity of mechanistic target of rapamycin complex 1 (mTORC1) governs the sensitivity of reserve ISCs to injury. CR inhibits mTORC1 in these cells, protecting them against DNA damage, while mTORC1 stimulation, either genetically or through nutrient sensing, sensitizes reserve ISCs to injury, thus compromising regeneration of the epithelium. These data delineate a critical role for mTORC1 in epithelial regeneration and inform clinical strategies based on nutrient modulation., Graphical Abstract, Highlights • Calorie restriction increases the radioresistant intestinal stem cell (ISC) pool • Calorie restriction suppresses mTORC1 activity in reserve ISCs • Reserve ISC-autonomous mTORC1 activity governs ISC survival post injury • Calorie-restricted reserve ISCs have enhanced regenerative capacity post injury, In this study, Yousefi et al. identify reserve intestinal stem cell-autonomous suppression of mTORC1 activity in response to calorie restriction as the basis for enhanced regeneration of the intestinal epithelium after DNA-damaging injury. Conversely, the authors demonstrate that acute nutrient-based stimulation of mTORC1 prior to injury results in reserve stem cell apoptosis and intestinal regenerative failure.

Intestinal regeneration and tumorigenesis are believed to be driven by intestinal stem cells (ISCs). Elucidating mechanisms underlying ISC activation during regeneration and tumorigenesis can help uncover the underlying principles of intestinal homeostasis and disease including colorectal cancer. Here we show that miR-31 drives ISC proliferation, and protects ISCs against apoptosis, both during homeostasis and regeneration in response to ionizing radiation injury. Furthermore, miR-31 has oncogenic properties, promoting intestinal tumorigenesis. Mechanistically, miR-31 acts to balance input from Wnt, BMP, TGFβ signals to coordinate control of intestinal homeostasis, regeneration and tumorigenesis. We further find that miR-31 is regulated by the STAT3 signaling pathway in response to radiation injury. These findings identify miR-31 as a critical modulator of ISC biology, and a potential therapeutic target for a broad range of intestinal regenerative disorders and cancers., eLife digest Cells lining the inner wall of the gut help to absorb nutrients and to protect the body against harmful microbes and substances. Being on the front line of defense means that these cells often sustain injuries. Specialized cells called intestinal stem cells keep the tissues healthy by replacing the damaged and dying cells. The intestinal stem cells can produce copies of themselves and generate precursors of the gut cells. They also have specific mechanism to protect themselves from cell death. These processes are regulated by different signals that are generated by the cell themselves or the neighboring cells. If these processes get out of control, cells can easily be depleted or develop into cancer cells. Until now, it remained unclear how intestinal stem cells can differentiate between and respond to multiple and simultaneous signals. It is known that short RNA molecules called microRNA play an important role in the signaling pathways of damaged cells and during cancer development. In the gut, different microRNAs, including microRNA-31,help to keep the gut lining intact. However, previous research has shown that bowel cancer cells also contain high amounts of microRNA-31. To see whether microRNA-31 plays a role in controlling the signaling systems in intestinal stem cells, Tian, Ma, Lv et al. looked at genetically modified mice that either had too much microRNA-31 or none. Mice with too much microRNA-31 produced more intestinal stem cells and were able to better repair any cell damage. Mice without microRNA-31 gave rise to fewer intestinal stem cellsand had no damage repair, but were able to stop cancer cells in the gut from growing. The results showed that microRNA-31 in intestinal stem cells helps the cells to divide and to protect themselves from cell death. It controlled and balanced the different types of cell signaling by either repressing or activating various signals. When Tian et al. damaged the stem cells using radiation, the cells increased their microRNA-31 levels as a defense mechanism. This helped the cells to survive and to activate repair mechanisms. Furthermore, Tian et al. discovered that microRNA-31 can enhance the growth of tumors. These results indicate that microRNA-31 plays an important role both in repairing gut linings and furthering tumor development. A next step will be to see whether cancer cells use microRNA-31 to protect themselves from chemo- and radiation therapy. This could help scientists find new ways to render cancerous cells more susceptible to existing cancer therapies.

Hair follicles (HFs) undergo precisely regulated cycles of active regeneration (anagen), involution (catagen), and relative quiescence (telogen). Hair follicle stem cells (HFSCs) play important roles in regenerative cycling. Elucidating mechanisms that govern HFSC behavior can help uncover the underlying principles of hair development, hair growth disorders, and skin cancers. RNA-binding proteins of the Musashi (Msi) have been implicated in the biology of different stem cell types, yet they have not been studied in HFSCs. Here we utilized gain- and loss-of-function mouse models to demonstrate that forced MSI2 expression retards anagen entry and consequently delays hair growth, whereas loss of Msi2 enhances hair regrowth. Furthermore, our findings show that Msi2 maintains quiescent state of HFSCs in the process of the telogen-to-anagen transition. At the molecular level, our unbiased transcriptome profiling shows that Msi2 represses Hedgehog signaling activity and that Shh is its direct target in the hair follicle. Taken together, our findings reveal the importance of Msi2 in suppressing hair regeneration and maintaining HFSC quiescence. The previously unreported Msi2-Shh-Gli1 pathway adds to the growing understanding of the complex network governing cyclic hair growth.

Summary The recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal., Graphical Abstract, Highlights • Proxy intestinal stem cell reporter alleles often mark heterogeneous populations • Discrepancies exist between proxy reporter activity and endogenous transcripts • Reserve and active intestinal stem cells are molecularly distinct • Reserve intestinal stem cells give rise to active stem cells during homeostasis, In this study, Lengner and colleagues utilize single-cell profiling to provide a comparative analysis of epithelial cells marked by a number of putative intestinal stem cell proxy reporter alleles. They confirm the existence of two molecularly distinct stem cell populations governing homeostasis, with a long-term reserve stem cell giving rise to a short-term active stem cell.

Musashi-2 is an important regulator of the hematopoietic stem cell translatome and balances HSC homeostasis and lineage bias., Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF-β signaling. Msi2-null HSCs are insensitive to TGF-β–mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias.

Background & Aims Levels of microRNA 31 (MIR31) are increased in intestinal tissues from patients with inflammatory bowel diseases and colitis-associated neoplasias. We investigated the effects of this microRNA on intestinal inflammation by studying mice with colitis. Methods We obtained colon biopsy samples from 82 patients with ulcerative colitis (UC), 79 patients with Crohn's disease (CD), and 34 healthy individuals (controls) at Shanghai Tenth People's Hospital. MIR31- knockout mice and mice with conditional disruption of Mir31 specifically in the intestinal epithelium (MIR31 conditional knockouts) were given dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. We performed chromatin immunoprecipitation and luciferase assays to study proteins that regulate expression of MIR31, including STAT3 and p65, in LOVO colorectal cancer cells and organoids derived from mouse colon cells. Partially hydrolyzed alpha-lactalbumin was used to generate peptosome nanoparticles, and MIR31 mimics were loaded onto their surface using electrostatic adsorption. Peptosome–MIR31 mimic particles were encapsulated into oxidized konjac glucomannan (OKGM) microspheres, which were administered by enema into the large intestines of mice with DSS-induced colitis. Intestinal tissues were collected and analyzed by histology and immunohistochemistry. Results Levels of MIR31 were increased in inflamed mucosa from patients with CD or UC, and from mice with colitis, compared with controls. STAT3 and nuclear factor-κB activated transcription of MIR31 in colorectal cancer cells and organoids in response to tumor necrosis factor and interleukin (IL)6. MIR31-knockout and conditional-knockout mice developed more severe colitis in response to DSS and TNBS, with increased immune responses, compared with control mice. MIR31 bound to 3ʹ untranslated regions of Il17ra and Il7r messenger RNAs (RNAs) (which encode receptors for the inflammatory cytokines IL17 and IL7) and Il6st mRNA (which encodes GP130, a cytokine signaling protein). These mRNAs and proteins were greater in MIR31-knockout mice with colitis, compared with control mice; MIR31 and MIR31 mimics inhibited their expression. MIR31 also promoted epithelial regeneration by regulating the WNT and Hippo signaling pathways. OKGM peptosome–MIR31 mimic microspheres localized to colonic epithelial cells in mice with colitis; they reduced the inflammatory response, increased body weight and colon length, and promoted epithelial cell proliferation. Conclusions MIR31, increased in colon tissues from patients with CD or UC, reduces the inflammatory response in colon epithelium of mice by preventing expression of inflammatory cytokine receptors (Il7R and Il17RA) and signaling proteins (GP130). MIR31 also regulates the WNT and Hippo signaling pathways to promote epithelial regeneration following injury. OKGM peptosome–MIR31 microspheres localize to the colon epithelium of mice to reduce features of colitis. Transcript Profiling: GSE123556

Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinson's disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear.Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base.Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.

The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters. Loss of P1-driven expression causes developmental defects with cleidocranial dysplasia-like syndromes that persist in the postnatal skeleton. P1 activity is robust in preosteogenic mesenchyme and at the onset of bone formation but decreases as bone matures. Homozygous Runx2-P1(lacZ/lacZ) mice have a normal life span but exhibit severe osteopenia and compromised bone repair in adult mice because of osteoblastic defects and not increased osteoclastic resorption. Gene expression profiles of bone, immunohistochemical studies, and ex vivo differentiation using calvarial osteoblasts and marrow stromal cells identified mechanisms for the skeletal phenotype. The findings indicate that P1 promoter activity is necessary for generating a threshold level of Runx2 protein to commit sufficient osteoprogenitor numbers for normal bone formation. P1 promoter function is not compensated via the P2 promoter. However, the P2 transcript with compensatory mechanisms from bone morphogenetic protein (BMP) and Wnt signaling is adequate for mineralization of the bone tissue that does form. We conclude that selective utilization of the P1 and P2 promoters enables the precise spatiotemporal expression of Runx2 necessary for normal skeletogenesis and the maintenance of bone mass in the adult.

Patients with dyskeratosis congenita (DC) suffer from stem cell failure in numerous organs, including the skin, intestine, blood, lung, and liver, due to hypomorphic loss-of-function mutations in proteins that promote telomere elongation or capping. Few therapeutic options exist for this disorder, and while patients are treated with bone marrow transplantation to restore hematopoietic function, this does not address the multi-organ failure, and in some cases can exacerbate it. We generated isogenic DC patient and disease allele-corrected induced pluripotent stem (iPS) cell lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction to model the stem cell failure characteristic of DC in multiple organ systems through the directed differentiation. We focused first on the intestinal epithelium, as GI manifestations of DC (enteropathy/enterocolitis ) are often the first and most severe symptoms in pediatric patients. We demonstrate that DC tissue has suboptimal Wnt pathway activity causing intestinal stem cell failure and that enhanced expression of the telomere-capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with the clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. These findings establish the existence of a feed-forward loop between telomerase/telomere capping and the canonical Wnt pathway which is broken in DC, but can be restored by promoting capping and/or Wnt pathway agonists. Moving forward, we have begun to extend these investigations into the lung and liver, where fibrosis and organ failure are major clinical concerns in DC. Thus, the isogenic DC iPS cell model provides a framework for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients. Disclosures No relevant conflicts of interest to declare.

SummaryThe presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen.

The promise of treating human genetic and degenerative diseases through the application of pluripotent cell-based tissue engineering and regenerative medicine has come significantly closer to realization since the isolation of human embryonic stem (ES) cells. While the study of ES cells has greatly increased our fundamental understanding of pluripotency, technical and ethical limitations have been seemingly insurmountable impediments to the application of these cells in the clinic. The recent discovery that somatic mammalian cells can be epigenetically reprogrammed to a pluripotent state through the exogenous expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC has yielded a new cell type for potential application in regenerative medicine, the induced pluripotent stem (iPS) cell. Here we discuss how advances in iPS cell technology have led to the generation of patient-specific cell lines that can potentially be used to model human diseases and ultimately act as therapeutic agents.

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

Since its discovery as a critical regulator of pluripotency in embryonic stem (ES) cells and the inner cells mass of the developing blastocyst, the Pou domain-containing transcription factor Oct4 has become a proxy for “stemness” in numerous studies of somatic stem cells as it’s presence is often taken as evidence of pluripotency in these cells. Recent studies, however, have demonstrated that not only is Oct4 dispensable for maintaining potency in somatic stem cell compartments, but also that the methods applied to detect Oct4 and the interpretation of the resulting data may be flawed. Here we contrast pathways known to govern pluripotency in embryonic stem cells with those in adult stem cells and critically discuss the concept of pluripotency in adult stem cells of the mammalian soma.

Summary Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues, including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation. We show that DC tissue has suboptimal Wnt pathway activity causing intestinal stem cell failure and that enhanced expression of the telomere-capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with the clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. Thus, the isogenic DC cell model provides a platform for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients.

Wnt pathway-driven proliferation and renewal of the intestinal epithelium must be tightly controlled to prevent development of cancer and barrier dysfunction. Although type I interferons (IFN) produced in the gut under the influence of microbiota are known for their antiproliferative effects, the role of these cytokines in regulating intestinal epithelial cell renewal is largely unknown. Here we report a novel role for IFN in the context of intestinal knockout of casein kinase 1α (CK1α), which controls the ubiquitination and degradation of both β-catenin and the IFNAR1 chain of the IFN receptor. Ablation of CK1α leads to the activation of both β-catenin and IFN pathways and prevents the unlimited proliferation of intestinal epithelial cells despite constitutive β-catenin activity. IFN signaling contributes to the activation of the p53 pathway and the appearance of apoptotic and senescence markers in the CK1α-deficient gut. Concurrent genetic ablation of CK1α and IFNAR1 leads to intestinal hyperplasia, robust attenuation of apoptosis, and rapid and lethal loss of barrier function. These data indicate that IFN play an important role in controlling the proliferation and function of the intestinal epithelium in the context of β-catenin activation.