产品说明

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

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system,neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay,that all neurospheres are derived from a NSC,and provided evidence that it overestimates NSC frequency,rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA),which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs,thereby facilitating further advances in the promising application of NSCs for therapeutic use. View Publication

BACKGROUND Ependymoma management remains challenging because of the inherent chemoresistance of this tumor. To determine whether ependymoma stem cells (SCs) might contribute to therapy resistance,we investigated the sensitivity of ependymoma SCs to temozolomide and etoposide. METHODS The efficacies of the two DNA damaging agents were explored in two ependymoma SC lines in vitro and in vivo models. RESULTS Ependymoma SC lines were highly sensitive to temozolomide and etoposide in vitro,but only temozolomide impaired tumor-initiation properties. Consistently,temozolomide but not etoposide showed significant antitumoral activity on ependymoma SC-driven subcutaneous and orthotopic xenografts by reducing the mitotic fraction. In vitro temozolomide at the EC50 (10 µM) induced accumulation of cells in the G2/M phase that was unexpectedly accompanied by downregulation of p27 and p21 without modulation of full-length p53 (FLp53). Differentiation-committed ependymoma SCs acquired resistance to temozolomide. Inhibition of proliferation was partly due to apoptosis,that occurred earlier in differentiated cells as compared to neurospheres. The activation of apoptosis correlated with an increase in p53β/γ isoforms without modulation of FLp53 under both serum-free and differentiation-promoting media. Incubation of cells in both conditions with temozolomide resulted in increased glioneuronal differentiation exhibiting elevated glial fibrillary acidic protein,galactosylceramidase,and βIII-tubulin expression compared to untreated controls. O(6)-methylguanine DNA methyltransferase (MGMT) transcript levels were very low in SCs,and were increased by treatment and,epigenetically,by differentiation through MGMT promoter unmethylation. CONCLUSION Ependymoma growth might be impaired by temozolomide through preferential depletion of a less differentiated,more tumorigenic,MGMT-negative cell population with stem-like properties. View Publication

Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types,they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial,skeletal,and neurological anomalies. Heterozygous females are more severely affected than hemizygous males,a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation,no direct evidence for this has been demonstrated. Here,by generating hiPSCs from CFNS patients,we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells,thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients. View Publication

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor (EPOR). The presence of EPO and its receptor in the CNS suggests a different function for EPO other than erythropoiesis. The purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation of neonatal spinal cord-derived neural progenitor cells. The effect of EPO on cell cycle progression was also examined,as well as the signaling cascades involved in this process. Our results showed that EPOR was present in the neural progenitor cells and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated neural progenitor cells indicated a reduced percentage of cells in G0/G1 phase,whereas the cell proliferation index (S phase plus G2/M phase) was increased. EPO also increased the proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells. With respect to the cell cycle signaling,we examined the cyclin-dependent kinases D1,D2 and E,and cyclin-dependent kinase inhibitors,p21cip1,p27kip1 and p57kip2. No significant differences were observed in the expression of these transcripts after EPO administration. Interestingly,the anti-apoptotic factors,mcl-1 and bcl-2 were significantly increased twofold. Moreover,these specific effects of EPO were eliminated by incubation of the progenitor cells with anti-EPO neutralizing antibody. Those observations suggested that EPO may play a role in normal spinal cord development by regulating cell proliferation and apoptosis. View Publication

OBJECTIVE Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines,suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique,here we report the characterization of GBM cell lines including GBM variants. METHODS Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. RESULTS Thirteen oncosphere lines derived from GBM and GBM variants,including a GBM with PNET features and a GBM with oligodendroglioma component,were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas,including invasion,necrosis,abnormal vascularization,and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. CONCLUSIONS We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study. 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

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

While no effective therapy is available for the treatment of methamphetamine (METH)-induced neurotoxicity,aerobic exercise is being proposed to improve depressive symptoms and substance abuse outcomes. The present study focuses on the effect of exercise on METH-induced aberrant neurogenesis in the hippocampal dentate gyrus in the context of the blood-brain barrier (BBB) pathology. Mice were administered with METH or saline by i.p. injections for 5 days with an escalating dose regimen. One set of mice was sacrificed 24 h post last injection of METH,and the remaining animals were either subjected to voluntary wheel running (exercised mice) or remained in sedentary housing (sedentary mice). METH administration decreased expression of tight junction (TJ) proteins and increased BBB permeability in the hippocampus. These changes were preserved post METH administration in sedentary mice and were associated with the development of significant aberrations of neural differentiation. Exercise protected against these effects by enhancing the protein expression of TJ proteins,stabilizing the BBB integrity,and enhancing the neural differentiation. In addition,exercise protected against METH-induced systemic increase in inflammatory cytokine levels. These results suggest that exercise can attenuate METH-induced neurotoxicity by protecting against the BBB disruption and related microenvironmental changes in the hippocampus. View Publication

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma,aging,and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein,we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging,propensity to form spheres,and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2,OCT4,LGR5,TP63 (p63),as well as neural crest marker genes PSIP1 (p75(NTR)),PAX3,SOX9,AP2B1 (AP-2β),and NES,generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2,β-III tubulin,and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion,we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. View Publication

Plp1 gene expression occurs very early in development,well before the onset of myelination,creating a conundrum with regard to the function of myelin proteolipid protein (PLP),one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity,we found that,at very early time points,PLP-EGFP was expressed in Sox2+ undifferentiated precursors in the spinal cord ventricular zone (VZ),as well as in the progenitors of both neuronal and glial lineages. As development progressed,most PLP-EGFP-expressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord,and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP,normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However,the orientation and length of OPC processes during migration was abnormal in Plp1-null mice,suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth. View Publication