技术资料

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4,Sox2,Klf4 and c-myc (OSKM) in the presence of sodium butyrate (1-3). We used this method to reprogram late passage (textgreaterp10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665,Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81,a surface marker of pluripotent cells,separated from non-reprogrammed fibroblasts and manually passaged (4,5). These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions,directly from the reprogramming plate. Starting from the first passage,hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4,as well as nuclear markers Oct3/4,Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals( 6),human newborn fibroblasts,as well as human keratinocytes. View Publication

Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism,digestion,satiety and lipid absorption,yet their development remains poorly understood. Here we show that Arx,a homeodomain-containing transcription factor,is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice,removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-,glucagon/GLP-1-,CCK-,secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition,depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK,secretin and glucagon while expression of a pan-intestinal epithelial marker,CDX2,and other non-endocrine markers remained unchanged. Taken together,our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations. View Publication

Neurogenesis persists in the rodent dentate gyrus (DG) throughout adulthood but declines with age and stress. Neural progenitor cells (NPCs) residing in the subgranular zone of the DG are regulated by an array of growth factors and respond to the microenvironment,adjusting their proliferation level to determine the rate of neurogenesis. Here we report that genetic deletion of neurofibromin (Nf1),a tumor suppressor with RAS-GAP activity,in adult NPCs enhanced DG proliferation and increased generation of new neurons in mice. Nf1 loss-associated neurogenesis had the functional effect of enhancing behavioral responses to subchronic antidepressants and,over time,led to spontaneous antidepressive-like behaviors. Thus,our findings establish an important role for the Nf1-Ras pathway in regulating adult hippocampal neurogenesis,and demonstrate that activation of adult NPCs is sufficient to modulate depression- and anxiety-like behaviors. View Publication

EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma,its expression in other tumor types remains controversial. In this study,we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII,but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial-mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically,EGFRvIII mediated its effects through the Wnt/β-catenin pathway,leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together,our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. View Publication

Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower,less toxic,doses. Experimentally,high doses induce rapid DNA damage and cytotoxicity,which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses,without causing immediate cytotoxicity,produce an antitumor memory" response� View Publication

The subunit genes encoding human chorionic gonadotropin,CGA,and CGB,are up-regulated in human trophoblast. However,they are effectively silenced in choriocarcinoma cells by ectopically expressed POU domain class 5 transcription factor 1 (POU5F1). Here we show that POU5F1 represses activity of the CGA promoter through its interactions with ETS2,a transcription factor required for both placental development and human chorionic gonadotropin subunit gene expression,by forming a complex that precludes ETS2 from interacting with the CGA promoter. Mutation of a POU5F1 binding site proximal to the ETS2 binding site does not alter the ability of POU5F1 to act as a repressor but causes a drop in basal promoter activity due to overlap with the binding site for DLX3. DLX3 has only a modest ability to raise basal CGA promoter activity,but its coexpression with ETS2 can up-regulate it 100-fold or more. The two factors form a complex,and both must bind to the promoter for the combination to be transcriptionally effective,a synergy compromised by POU5F1. Similarly,in human embryonic stem cells,which express ETS2 but not CGA,ETS2 does not occupy its binding site on the CGA promoter but is found instead as a soluble complex with POU5F1. When human embryonic stem cells differentiate in response to bone morphogenetic protein-4 and concentrations of POU5F1 fall and hCG and DLX3 rise,ETS2 then occupies its binding site on the CGA promoter. Hence,a squelching mechanism underpins the transcriptional silencing of CGA by POU5F1 and could have general relevance to how pluripotency is maintained and how the trophoblast lineage emerges from pluripotent precursor cells. View Publication

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet,taken up by cells and tissues,and subsequently reduced to dihydrolipoic acid (DHLA). In view of the recent application of DHLA as a hydrophilic nanomaterial preparation,determination of its biosafety profile is essential. In the current study,we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage,subsequent embryonic attachment and outgrowth in vitro,in vivo implantation by embryo transfer,and early embryonic development in an animal model. Blastocysts treated with 50 μM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably,the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover,in vitro treatment with 50 μM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 μM DHLA led to decreased early embryo development,specifically,inhibition of development to the blastocyst stage. However,it appears that concentrations of DHLA lower than 50 μM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 μM DHLA induces apoptosis and retards early pre- and post-implantation development,and support the potential of DHLA to induce embryonic cytotoxicity. View Publication

Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib,nilotinib and dasatinib are currently used to treat CML,but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML,but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl,which explains the inhibitor's activity against several imatinib-resistant mutants,and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name bosutinib"� View Publication

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits,a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain,containing neural stem cells with brain repair potential. Accordingly,the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation,evaluated by BrdU incorporation assay,in the neuroblast lineage. Moreover,by using a cell viability assay (TUNEL) we found that,in contrast to AMPA,CX546 did not cause cell death. Also,both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly,short exposure to CX546 increased axonogenesis,as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK),and dendritogenesis (MAP2-positive neurites). Altogether,this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies. View Publication

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However,research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly,this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose. View Publication

Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end,we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption,but in parallel functional integration of all probes mandatory for process monitoring,that is,for pO₂ and pH,experiments were performed in 100 mL culture volume in a mini reactor platform" consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×10�?� hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained� View Publication

Monocytes and macrophages are often claimed to have limited potential for proliferation in vivo and in vitro although a human monocyte subset with increased potential to proliferate in culture,termed the proliferative monocyte (PM),has previously been identified. The response of the putatively less mature PM to conditions conducive to haematopoietic stem cell culture was determined. Co-culture of monocytes on a HUVEC monolayer induced up to four cell divisions in a 9 day period. The PM response to haematopoietic growth factors (Flt3L,SCF,IL-6,IL-3 and M-CSF) was determined. M-CSF induced the greatest proliferative response in PM; IL-3 and Flt3L reduced basal and M-CSF-induced proliferation. The inhibition of M-CSFR kinase activity by GW2580 indicated that the ligand(s) for this receptor was a potent inducer of proliferation of this subset; inhibitors of intracellular signalling pathways also reduced PM proliferation. View Publication