技术资料

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors,most notably HIF2α (also known as EPAS1),are expressed in and required for maintenance of cancer stem cells (CSCs). However,the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex,displaces VHL-associated Cullin 2,and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma,ID2 positively modulates HIF2α activity. Conversely,elevated expression of DYRK1 phosphorylates Thr27 of ID2,leading to HIF2α destabilization,loss of glioma stemness,inhibition of tumour growth,and a more favourable outcome for patients with glioblastoma. View Publication

The existence of a hyaluronic acid-rich node and duct system (HAR-NDS) within the lymphatic and blood vessels was demonstrated previously. The HAR-NDS was enriched with small (3.0-5.0 μm in diameter),adult stem cells with properties similar to those of the very small embryonic-like stem cells (VSELs). Sca-1(+)Lin(-)CD45(-) cells were enriched approximately 100-fold in the intravascular HAR-NDS compared with the bone marrow. We named these adult stem cells node and duct stem cells (NDSCs)." NDSCs formed colonies on C2C12 feeder layers were positive for fetal alkaline phosphatase and could be subcultured on the feeder layers. NDSCs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(+) while VSELs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(-). NDSCs had higher sphere-forming efficiency and proliferative potential than VSELs and they were found to differentiate into neuronal cells in vitro. Injection of NDSCs into mice partially repaired ischemic brain damage. Thus we report the discovery of potential adult stem cells that may be involved in tissue regeneration. The intravascular HAR-NDS may serve as a route that delivers these stem cells to their target tissues. View Publication

A number of genes regulate regeneration of peripheral axons,but their ability to drive axon growth and regeneration in the central nervous system (CNS) remains largely untested. To address this question we overexpressed eight transcription factors and one small GTPase alone and in pairwise combinations to test whether combinatorial overexpression would have a synergistic impact on CNS neuron neurite growth. The Jun oncogene/signal transducer and activator of transcription 6 (JUN/STAT6) combination increased neurite growth in dissociated cortical neurons and in injured cortical slices. In injured cortical slices,JUN overexpression increased axon growth to a similar extent as JUN and STAT6 together. Interestingly,JUN overexpression was not associated with increased growth associated protein 43 (GAP43) or integrin alpha 7 (ITGA7) expression,though these are predicted transcriptional targets. This study demonstrates that JUN overexpression in cortical neurons stimulates axon growth,but does so independently of changes in expression of genes thought to be critical for JUNs effects on axon growth. We conclude that JUN activity underlies this CNS axonal growth response,and that it is mechanistically distinct from peripheral regeneration responses,in which increases in JUN expression coincide with increases in GAP43 expression. View Publication

Inflammatory activation precedes and correlates with accumulating τ lesions in Alzheimer's disease and tauopathies. However,the relationship between neuroinflammation and etiology of pathologic τ remains elusive. To evaluate whether inflammatory signaling may promote or accelerate neurofibrillary tangle pathology,we explored the effect of recombinant adeno-associated virus (rAAV)-mediated overexpression of a master inflammatory cytokine,IFN-γ,on τ phosphorylation. In initial studies in primary neuroglial cultures,rAAV-mediated expression of IFN-γ did not alter endogenous τ production or paired helical filament τ phosphorylation. Next,we tested the effect of rAAV-mediated expression of IFN-γ in the brains of 2 mouse models of tauopathy: JNPL3 and rTg4510. In both models,IFN-γ increased 1) signal transducer and activator of transcription 1 levels and gliosis,and 2) hyperphosphorylation and conformational alterations of soluble τ compared with control cohorts. However,sarkosyl-insoluble phosphorylated τ levels and ubiquitin staining were unaltered in the IFN-γ cohorts. Notably,IFN-γ-induced τ hyperphosphorylation was associated with release of the inhibitory effect of glycogen synthase kinase 3β function by decreasing Ser9 phosphorylation. Our data suggest that type II IFN signaling can promote τ phosphorylation by modulating cellular kinase activity,though this is insufficient in accelerating neuritic tangle pathology. View Publication

Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis,which is associated with cell autophagy. However,the mechanism by which autophagy regulates neurogenesis remains undefined. Here,we show that Eva1a/Tmem166,an autophagy-related gene,regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons,both in vivo and in vitro. Conversely,overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover,Eva1a depletion activated the PIK3CA-AKT axis,leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore,addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion,suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively,these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation. View Publication

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods,however,cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (˜50 days,10 passages tested,accumulative ˜>10(10)-fold expansion) with both high growth rate (˜20-fold expansion/7 days) and high volumetric yield (˜2.0%A-%10(7)%cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient,affordable glioblastoma TICs for drug discovery. View Publication

In this study,we investigated the impact of Nardosinone,a bioactive component in Nardostachys root,on the proliferation and differentiation of neural stem cells. The neural stem cells were isolated from cerebrums of embryonic day 14 CD1 mice. The proliferation of cells was monitored using the cell counting kit-8 assay,bromodeoxyuridine incorporation and cell cycle analysis. Cell migration and differentiation were investigated with the neurosphere assay and cell specific markers,respectively. The results showed that Nardosinone promotes cells proliferation and increases cells migration distance in a dose-dependent manner. Nardosinone also induces the selective differentiation of neural stem cells to neurons and oligodendrocytes,as indicated by the expression of microtubule-associated protein-2 and myelin basic protein,respectively. Nardosinone also increases the expression of phospho-extracellular signal-regulated kinase and phospho-cAMP response element binding protein during proliferation and differentiation. In conclusion,this study reveals the regulatory effects of Nardosinone on neural stem cells,which may have significant implications for the treatment of brain injury and neurodegenerative diseases. View Publication

Giant cell tumor of bone (GCTB) is a very rare tumor entity,which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis,emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells,stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation,differentiation,migration,MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,immunohistochemistry,antibody protein array,Alu in situ hybridization,FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies,differentiated to osteoblasts,migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4,indicating self-renewal,invasion and differentiation potential. Compared with adherent-growing cells,markers for pluripotency,stemness and cancer progression,including the CSC surface marker c-Met,were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib,an inhibitor of c-Met in phase II trials,eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met(+) tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB. View Publication

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions,a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity migratory potential and a switch in morphology to a fibroblastoid phenotype as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors triptolide downregulated markers of CSCs proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA. View Publication

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples,while requiring low cell numbers. To date,a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative,EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris,doublets and dead cells from the analysis. For validation,the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers,HBEC recovered from BAL (2.3% of live cells),BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed,validated,and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. View Publication

Therapeutic modulation of phosphatidylinositol 3-kinase (PI3K)/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this,we created Olig2-cre:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy,chronic neurodegeneration,and death by 9 months. In contrast,during immediate postnatal development,oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Pten(fl/fl) mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor,NVP-BKM120,blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary,our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However,such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development. View Publication

Aberrant neural progenitor cell (NPC) proliferation and self-renewal have been linked to age-related neurodegeneration and neurodegenerative disorders including Alzheimer's disease (AD). Rhizoma Acori tatarinowii is a traditional Chinese herbal medicine against cognitive decline. In this study,we found that the extract of Rhizoma Acori tatarinowii (AT) and its active constituents,asarones,promote NPC proliferation. Oral administration of AT enhanced NPC proliferation and neurogenesis in the hippocampi of adult and aged mice as well as that of transgenic AD model mice. AT and its fractions also enhanced the proliferation of NPCs cultured in vitro. Further analysis identified α-asarone and β-asarone as the two active constituents of AT in promoting neurogenesis. Our mechanistic study revealed that AT and asarones activated extracellular signal-regulated kinase (ERK) but not Akt,two critical kinase cascades for neurogenesis. Consistently,the inhibition of ERK activities effectively blocked the enhancement of NPC proliferation by AT or asarones. Our findings suggest that AT and asarones,which can be orally administrated,could serve as preventive and regenerative therapeutic agents to promote neurogenesis against age-related neurodegeneration and neurodegenerative disorders. View Publication