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Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage tracing studies identified a subset of EECs that reside at +4 position for more than 2 weeks,most of which were BrdU-label-retaining cells. Under basal conditions,cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally,the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis,stem cell dynamics of the intestinal epithelium and novel insight in the development of EC-derived small intestinal tumors. View Publication

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure,the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches,the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling,and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice,the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked,demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens,inflammatory stimuli and aging can modify M cell-density in the gut,these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice,resulting in shortened survival times and increased disease susceptibility,equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection,whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases. View Publication

PURPOSE: Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells,which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. MATERIALS AND METHODS: The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1(high) and ALDH1(low) cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. RESULTS: ALDH1(high) cells were detected in 6.8% of 22Rv1 cells,which showed significantly higher tumorigenicity than ALDH1(low) cells in NOD/SCID mice (p textless 0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1(high) cells than in ALDH1(low) cells. ALDH1(high) cells also showed higher invasive capability and sphere forming capability than ALDH1(low) cells. CONCLUSIONS: Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1(high) population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay. View Publication

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here,we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS) drives cell-autonomous expression of type I cytokine receptor complexes (IL2r?–IL4r? and IL2r?–IL13r?1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS and Th2 cells producing IL4 and IL13. Activated IL2r?–IL4r? and IL2r?–IL13r?1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic,chromatin occupancy,and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus,paracrine signaling in the tumor microenvironment plays a key role in the KRAS-driven metabolic reprogramming of PDAC. SIGNIFICANCE: Type II cytokines,secreted by Th2 cells in the tumor microenvironment,can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions,providing candidate therapeutic targets in the KRAS pathway for this intractable disease. View Publication

p40,a Lactobacillus rhamnosus GG (LGG)-derived protein,transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells,leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation,this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells,which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfr(fl/fl),but not Egfr(fl/fl)-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells,exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA(+) cells and IgA production,which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells,fecal IgA levels,IgA(+)B220(+),IgA(+)CD19(+),and IgA(+) plasma cells in lamina propria of Egfr(fl/fl),but not of Egfr(fl/fl)-Vil-Cre,mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells,which may contribute to promoting IgA production. View Publication

The intestinal epithelial repair after injury is coordinated by intestinal stem cells (ISCs). Fucosylation catalyzed by fucosyltransferase 2 (FUT2) of the intestinal epithelium is beneficial to mucosal healing but poorly defined is the influence on ISCs. The dextran sulfate sodium (DSS) and lipopolysaccharide (LPS) model were used to assess the role of FUT2 on ISCs after injury. The apoptosis,function,and stemness of ISCs were analyzed using intestinal organoids from WT and Fut2?ISC (ISC-specific Fut2 knockout) mice incubated with LPS and fucose. N-glycoproteomics,UEA-1 chromatography,and site-directed mutagenesis were monitored to dissect the regulatory mechanism,identify the target fucosylated protein and the corresponding modification site. Fucose could alleviate intestinal epithelial damage via upregulating FUT2 and ?-1,2-fucosylation of ISCs. Oxidative stress,mitochondrial dysfunction,and cell apoptosis were impeded by fucose. Meanwhile,fucose sustained the growth and proliferation capacity of intestinal organoids treated with LPS. Contrarily,FUT2 depletion in ISCs aggravated the epithelial damage and disrupted the growth and proliferation capacity of ISCs via escalating LPS-induced endoplasmic reticulum (ER) stress and initiating the IRE1/TRAF2/ASK1/JNK branch of unfolded protein response (UPR). Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine (Asn) 862 mediated by FUT2 was identified to facilitate ISCs survival and self-renewal,and improve ISCs resistance to ER stress and inflammatory injury. Our study highlights a fucosylation-dependent protective mechanism of ISCs against inflammation,which may provide a fascinating strategy for treating intestinal injury disorders. View Publication

Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus,inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study,we investigated the effects of imetelstat (GRN163L),a potent telomerase inhibitor,on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro,telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally,imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat,but not control oligonucleotides,also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after textless4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice,concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat,suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. View Publication

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease,and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However,whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here,using intestinal organoids as a model of tissue regeneration,we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes,impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease. View Publication

To better characterize biological responses to atmospheric organic aerosols,the efficient delivery of aerosol to in vitro lung cells is necessary. In this study,chamber generated secondary organic aerosol (SOA) entered the commercialized exposure chamber (CULTEX® Radial Flow System Compact) where it interfaced with an electrostatic precipitator (ESP) (CULTEX® Electrical Deposition Device) and then deposited on a particle collection plate. This plate contained human lung cells (BEAS-2B) that were cultured on a membrane insert to produce an air-liquid interface (ALI). To augment in vitro assessment using the ESP exposure device,the particle dose was predicted for various sampling parameters such as particle size,ESP deposition voltage,and sampling flowrate. The dose model was evaluated against the experimental measured mass of collected airborne particles. The high flowrate used in this study increased aerosol dose but failed to achieve cell stability. For example,RNA in the ALI BEAS-2B cells in vitro was stable at 0.15 L/minute but decayed at high flowrates. The ESP device and the resulting model were applied to in vitro studies (i.e.,viability and IL-8 expression) of toluene SOA using ALI BEAS-2B cells with a flowrate of 0.15 L/minute,and no cellular RNA decay occurred. View Publication

Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However,limited understanding of human airway patterning has made this goal challenging. Here,we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach,we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases. View Publication

Focal adhesion kinase (FAK) has been implicated in the development of cancers,including those of the breast. Nevertheless,the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here,we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers,including aldehyde dehydrogenase,CD24,CD29,and CD61,we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their self-renewal and migration in vitro. Finally,through transplantation in NOD-SCID mice,we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together,these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. View Publication