参考文献

We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic,HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8-10 weeks for the production of functional mature motoneurons. In comparison with other methods,our protocol does not use feeder cells,has a minimum dependence on proteins (purmorphamine replacing SHH),has controllable adherent selection and is adaptable for scalable suspension culture. View Publication

OBJECTIVE: Bone marrow-derived mononuclear cells (BMCs) improve the functional recovery after ischemia. However,BMCs comprise a heterogeneous mixture of cells,and it is not known which cell types are responsible for the induction of neovascularization after cell therapy. Because cell recruitment is critically dependent on the expression of the SDF-1-receptor CXCR4,we examined whether the expression of CXCR4 may identify a therapeutically active population of BMCs. METHODS AND RESULTS: Human CXCR4(+) and CXCR4(-) BMCs were sorted by magnetic beads. CXCR4(+) BMCs showed a significantly higher invasion capacity under basal conditions and after SDF-1 stimulation. Hematopoietic or mesenchymal colony-forming capacity did not differ between CXCR4(+) and CXCR4(-) BMCs. Injection of CXCR4(+) BMCs in mice after induction of hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4(-) BMCs. Likewise,capillary density was significantly increased in CXCR4(+) BMC-treated mice. Because part of the beneficial effects of cell therapy were attributed to the release of paracrine effectors,we analyzed BMC supernatants for secreted factors. Importantly,supernatants of CXCR4(+) BMCs were enriched in the proangiogenic cytokines HGF and PDGF-BB. CONCLUSIONS: CXCR4(+) BMCs exhibit an increased therapeutic potential for blood flow recovery after acute ischemia. Mechanistically,their higher migratory capacity and their increased release of paracrine factors may contribute to enhanced tissue repair. View Publication

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections impair plasmacytoid dendritic cell (PDC) and natural killer (NK) cell subset numbers and functions,though little is known about PDC-NK cell interactions during these infections. We evaluated PDC-dependent NK cell killing and gamma interferon (IFN-gamma) and granzyme B production,using peripheral blood mononuclear cell (PBMC)-based and purified cell assays of samples from HCV- and HIV-infected subjects. CpG-enhanced PBMC killing and IFN-gamma and granzyme B activity (dependent on PDC and NK cells) were impaired in viremic HIV infection. In purified PDC-NK cell culture experiments,CpG-enhanced,PDC-dependent NK cell activity was cell contact and IFN-alpha dependent,and this activity was impaired in viremic HIV infection but not in HCV infection. In heterologous PDC-NK cell assays,impaired PDC-NK cell killing activity was largely attributable to an NK cell defect,while impaired PDC-NK cell IFN-gamma-producing activity was attributable to both PDC and NK cell defects. Additionally,the response of NK cells to direct IFN-alpha stimulation was defective in viremic HIV infection,and this defect was not attributable to diminished IFN-alpha receptor expression,though IFN-alpha receptor and NKP30 expression was closely associated with killer activity in viremic HIV infection but not in healthy controls. These data indicate that during uncontrolled HIV infection,PDC-dependent NK cell function is impaired,which is in large part attributable to defective IFN-alpha-induced NK cell activity and not to altered IFN-alpha receptor,NKP30,NKP44,NKP46,or NKG2D expression. View Publication

Mammalian cells generally require both mitogens and anchorage signals in order to proliferate. An important characteristic of many tumour cells is that they have lost this anchorage-dependent cell-cycle checkpoint,allowing them to proliferate without signals provided by their normal microenvironment. In the absence of anchorage signals from the extracellular matrix,many cell types arrest cell-cycle progression in G1 phase as a result of Rb-dependent checkpoints. However,despite inactivation of p53 and Rb proteins,SV40LT-expressing cells retain anchorage dependency,suggesting the presence of an uncharacterised cell-cycle checkpoint,which can be overridden by coexpression of oncogenic Ras. We report here that,although cyclin-CDK complexes persisted in suspension,proliferation was inhibited in LT-expressing cells by the CDK inhibitor p27(Kip1) (p27). Interestingly,this did not induce a stable arrest,but aberrant cell-cycle progression associated with stalled DNA replication,rereplication and chromosomal instability,which was sufficient to increase the frequency of oncogenic transformation. These results firstly indicate loss of anchorage in Rb- and p53-deficient cells as a novel mechanism for promotion of genomic instability; secondly suggest that anchorage checkpoints that protect normal cells from inappropriate proliferation act deleteriously in Rb- and p53-deficient cells to promote tumourigenesis; and thirdly indicate caution in the use of CDK inhibitors for cancer treatment. View Publication

OBJECTIVES Histone deacetylase (HDAC) is an important therapeutic target in cancer. Two of the main anticancer mechanisms of HDAC inhibitors are induction of terminal differentiation and inhibition of cell proliferation. To investigate the role of HDAC in maintenance of self-renewal and cell proliferation,we treated mesenchymal stem cells (MSCs) that originated from adipose tissue or umbilical cord blood with valproic acid (VPA) and sodium butyrate (NaBu). MATERIALS AND METHODS Human MSCs were isolated from mammary fat tissue and cord blood. We performed MTT assay and flow cytometry-based cell cycle analysis to assess self-renewal of MSCs. In vitro differentiation assays into osteogenic,adipogenic,neurogenic and chondrogenic lineages were conducted to investigate MSC multipotency. Immunocytochemistry,Western blot and reverse transcription-polymerase chain reaction were used to interrogate molecular pathways. RESULTS VPA and NaBu flattened the morphology of MSCs and inhibited their growth. VPA and NaBu activated the transcription of p21(CIP1/WAF1) by increasing the acetylation of histone H3 and H4 and eventually blocked the cell cycle at G2/M phase. The expression level of p16(INK4A),a cdk inhibitor that is closely related to cellular senescence,was not changed by HDAC inhibitor treatment. We performed controlled differentiation into bone,fat,cartilage and nervous tissue to elucidate the role of HDAC in the pluripotency of MSC to differentiate into functional tissues. VPA and NaBu decreased the efficiency of adipogenic,chondrogenic,and neurogenic differentiation as visualized by specific staining and reverse transcription-polymerase chain reaction. In contrast,osteogenic differentiation was elevated by HDAC inhibitor treatment. CONCLUSION HDAC activity is essential for maintaining the self-renewal and pluripotency of MSCs. View Publication

Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function,but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription,dynamic chromatin architecture,transcription factor binding,and genomic organization in regulation of erythrocyte membrane protein genes,we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly,most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites,located primarily in introns. Cooccupancy with FOG-1,SCL,and MTA-2 was found at all regions of GATA-1 binding,with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4,GATA-1,NF-E2,FOG-1,SCL,and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. View Publication

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes,indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model,transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (ptextless0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition,transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease. View Publication

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for textgreater10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media,the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR),HEScGRO,and KnockOut media,mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions,cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4,stage-specific embryonic antigens 4 (SSEA-4),and Tra-1-60. In addition,hESC maintained the expression of podocalyxin-like protein-1 (PODXL),an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills textgreater90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies,derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal,endodermal,and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%,higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications. View Publication

IL-33,a new member of the IL-1 family,has been described as an important inducer of Th2 cytokines and mediator of inflammatory responses. In this study,we demonstrate that murine basophils sorted directly from the bone marrow,without prior exposure to IL-3 or Fc(epsilon)R cross-linking,respond to IL-33 alone by producing substantial amounts of histamine,IL-4,and IL-6. These cells express ST2 constitutively and generate a cytokine profile that differs from their IL-3-induced counterpart by a preferential production of IL-6. In vivo,IL-33 promotes basophil expansion in the bone marrow (BM) through an indirect mechanism of action depending on signaling through the beta(c) chain shared by receptors for IL-3,GM-CSF,and IL-5. IL-3 can still signal through its specific beta(IL-3) chain in these mutant mice,which implies that it is not the unique growth-promoting mediator in this setup,but requires IL-5 and/or GMCSF. Our results support a major role of the latter growth factor,which is readily generated by total BM cells as well as sorted basophils in response to IL-33 along with low amounts of IL-3. Furthermore,GM-CSF amplifies IL-3-induced differentiation of basophils from BM cells,whereas IL-5 that is also generated in vivo,affects neither their functions nor their growth in vitro or in vivo. In conclusion,our data provide the first evidence that IL-33 not only activates unprimed basophils directly,but also promotes their expansion in vivo through induction of GM-CSF and IL-3. View Publication

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication