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The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular,this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells),hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here,we described a new platform which enables,rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular,we devised a protocol that,combining the expression of the CRISPR components with neurogenic factors,generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy,fast and that does not require the generation of stable isogenic clones,practice that is time consuming and for some genes not feasible. View Publication

UNLABELLED: Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors. INTRODUCTION: Imatinib mesylate,an orally active inhibitor of the c-abl,c-kit,and platelet-derived growth factor receptor (PDGFR) tyrosine kinases,is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia,suggesting that imatinib might have adverse effects on the skeleton. However,biochemical markers of bone formation increase in patients with CML starting imatinib therapy,whereas bone resorption is unchanged,despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients. MATERIALS AND METHODS: Osteoblast differentiation was assessed using a mineralization assay,proliferation by [(3)H]thymidine incorporation,and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples,osteoblastic cells,and murine bone marrow cultures. Osteoprotegerin was measured by ELISA. RESULTS: The molecular targets of imatinib are expressed in bone cells. In vitro,imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures,imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells. CONCLUSIONS: Taken together with recent in vivo data,these results suggest a role for the molecular targets of imatinib in bone cell function,that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation,and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors. View Publication

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields,cellular heterogeneity,and limited scalability. In the present study,we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes,secreted hepatic proteins such as Albumin,Alpha Fetoprotein,and Fibrinogen,metabolized ammonia,and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes,such as LDL storage and uptake,ICG uptake and release,and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure,providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. View Publication

In fura-2-loaded ECV304 cells ionomycin elicited a saturable biphasic change in intracellular Ca2+ concentration ([Ca2+]i),where the initial phase represented mobilization of intracellular stores and the sustained component represented Ca2+ influx. To examine whether ionomycin could stimulate influx via a store-dependent mechanism. Mn2+ entry was monitored by the quenching of fura-2 fluorescence: influx was enhanced even after ionomycin wash-out,provided that internal stores were not refilled with Ca2+. Moreover,the maximal rate of histamine-stimulated Mn2+ entry was unaffected by ionomycin,suggesting a common route of entry. The Ca(2+)-entry blocker SK&F 96365 inhibited both the ionomycin-induced Mn2+ entry and the sustained [Ca2+]i response to the ionophore (leaving the initial peak [Ca2+]i response unaffected). In other experiments,although addition of ionomycin further increased the plateau phase induced by 100 microM histamine,the increase was completely abolished by pretreatment with the store Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). Furthermore,in store-depleted cells,re-addition of 1 mM extracellular Ca2+ (in the presence of CPA plus histamine) led to a rapid rise in [Ca2+]i,dependent on Ca2+ influx,with kinetics that were not enhanced by ionomycin. These data suggest that ionomycin acts primarily at the level of the internal Ca2+ stores,so that,at the concentrations used here (textless or = 1 microM),it increases Ca2+ (and Mn2+) influx via activation of endogenous entry pathways and not by plasmalemmal translocation. View Publication

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. View Publication

The immunosuppressant,rapamycin,inhibits cell growth by interfering with the function of a novel kinase,termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein,PHAS-1,in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line,which was selected for resistance to the growth-inhibitory action of rapamycin,was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast,the PI 3-kinase inhibitor,wortmannin,reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM),wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor,LY294002,at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR,and potentially those of other high molecular weight PI 3-kinase homologs,are directly affected by cellular treatment with wortmannin or LY294002. View Publication