技术资料

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication

Glycogen synthase kinase 3 (GSK-3) functions in the regulation of glycogen metabolism,in the cell cycle,and in immune responses and is targeted by some viruses to favor the viral life cycle. Inhibition of GSK-3 by 6-bromoindirubin-3'-acetoxime (BIO-acetoxime),a synthetic derivative of a compound from the Mediterranean mollusk Hexaplex trunculus,protects cells from varicella infection. In this study,we examined the effects of BIO-acetoxime against herpes simplex virus-1 (HSV-1) infection in human oral epithelial cells,which represent a natural target cell type. The results revealed that BIO-acetoxime relieves HSV-1-induced cytopathic effects and apoptosis. We also found that BIO-acetoxime reduced viral yields and the expression of different classes of viral proteins. Furthermore,addition of BIO-acetoxime before,simultaneously with or after HSV-1 infection significantly reduced viral yields. Collectively,BIO-acetoxime may suppress viral gene expression and protect oral epithelial cells from HSV-1 infection. These results suggest the possible involvement of GSK-3 in HSV-1 infection. View Publication

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus,hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these,37 hiPSC lines were generated from fetal hepatocytes,2 hiPSC lines from normal hepatocytes,and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome,type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression,flow cytometry,immunocytochemistry,and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors,while fetal hepatocytes could be reprogrammed with three (OCT4,SOX2,NANOG) or four factors (OCT4,SOX2,NANOG,LIN28 or OCT4,SOX2,KLF4,C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes,although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation. View Publication

Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze depalmitoylation of membrane-anchored,palmitoylated H-Ras and growth-associated protein-43 (GAP-43),respectively. However,the mechanism(s) of cytosol-membrane shuttling of APT1 and APT2,required for depalmitoylating their substrates H-Ras and GAP-43,respectively,remained largely unknown. Here,we report that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover,blocking palmitoylation adversely affects membrane localization of both APT1 and APT2 and that of their substrates. We also demonstrate that APT1 not only catalyzes its own depalmitoylation but also that of APT2 promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both thioesterases. Furthermore,shRNA suppression of APT1 expression or inhibition of its thioesterase activity by palmostatin B markedly increased membrane localization of APT2,and shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. In addition,mutagenesis of the active site Ser residue to Ala (S119A),which renders catalytic inactivation of APT1,also increased its membrane localization. Taken together,our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates,facilitating steady-state membrane localization and function of both. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Proplatelet formation is a part of the intricate process by which platelets are generated by their precursor cell,the megakaryocyte. The processes that drive megakaryocyte maturation and platelet production are however still not well understood. The protein kinase C (PKC) family of serine/threonine kinases has been demonstrated as an important regulator of megakaryocyte maturation and proplatelet formation,but little investigation has been made on the individual isoforms. We have previously shown,in mouse models,that PKCα plays a vital role in regulating platelet function,so in this study we aimed to investigate the role of PKCα in megakaryocyte function using the same Prkca(-)(/)(-) mice. We assessed the role of global PKC and specifically PKCα in proplatelet formation in vitro,analyzed polyploidy in Prkca(-)(/)(-)-derived megakaryocytes and followed platelet recovery in platelet-depleted Prkca(-)(/)(-) mice. We show reduced proplatelet formation in the presence of global PKC blockade. However,in the presence of a selective classical PKC isoform inhibitor,Go6976,proplatelet formation was conversely enhanced. PKCα null megakaryocytes also showed enhanced proplatelet formation,as well as a shift to greater polyploidy. In vivo,platelet production was enhanced in response to experimentally induced immune thrombocytopenia. In conclusion,our data indicate that classical PKC isoforms,and more specifically PKCα,are negative regulators of proplatelet formation. PKCα appears to negatively regulate endomitosis,with the enhanced polyploidy observed in Prkca(-)(/)(-)-derived megakaryocytes. In vivo,these observations may culminate in the observed ability of Prkca(-)(/)(-) mice to recover more rapidly from a thrombocytopenic insult. View Publication

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However,transplantation of pig organs results in an acute immune rejection,leading to death of the organ within minutes. The $\$-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection,yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g.,gene targeting) that often require highly robust and proliferative cells. In this report,we generated GALT-KO piPSCs by the overexpression of POU5F1,SOX2,NANOG,LIN28,KLF-4,and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics,expressing integrated reprogramming genes in addition to the pluripotent markers AP,SSEA1,and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation,somatic cell nuclear transfer,or chimera formation. View Publication

Within the two neurogenic niches of the adult mammalian brain,i.e.,the subventricular zone lining the lateral ventricle and the subgranular zone of the hippocampus,there exist distinct populations of proliferating neural precursor cells that differentiate to generate new neurons. Numerous studies have suggested that epigenetic regulation by histone-modifying proteins is important in guiding precursor differentiation during development; however,the role of these proteins in regulating neural precursor activity in the adult neurogenic niches remains poorly understood. Here we examine the role of an NAD(+) -dependent histone deacetylase,SIRT1,in modulating the neurogenic potential of neural precursors in the neurogenic niches of the adult mouse brain. We show that SIRT1 is expressed by proliferating adult subventricular zone and hippocampal neural precursors,although its transcript and protein levels are dramatically reduced during neural precursor differentiation. Utilizing a lentiviral-mediated delivery strategy,we demonstrate that abrogation of SIRT1 signaling by RNAi does not affect neural precursor numbers or their proliferation. However,SIRT1 knock down results in a significant increase in neuronal production in both the subventricular zone and the hippocampus. In contrast,enhancing SIRT1 signaling either through lentiviral-mediated SIRT1 overexpression or through use of the SIRT1 chemical activator Resveratrol prevents adult neural precursors from differentiating into neurons. Importantly,knock down of SIRT1 in hippocampal precursors in vivo,either through RNAi or through genetic ablation,promotes their neurogenic potential. These findings highlight SIRT1 signaling as a negative regulator of neuronal differentiation of adult subventricular zone and hippocampal neural precursors. textcopyright 2013 Wiley Periodicals,Inc. View Publication

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells,including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy,we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs,we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells,and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Bone remodeling,a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs),is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature,osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence,anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²�?�/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones,along with significantly more OBs and fewer multinuclear OCs. In vitro,although Camkk2�?�/�?� mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs,bone marrow cells from Camkk2�?�/�?� mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective,cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further,we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand,genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target,nuclear factor of activated T cells,cytoplasmic (NFATc1). Moreover,in vivo administration of STO-609 results in increased OBs and diminished OCs,conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall,our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis. View Publication