参考文献

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors,octamer-binding transcription factor 4 (OCT4),(sex determining region Y)-box 2 (SOX2),Kruppel-like factor 4 (KLF4),and v-myc myelocytomatosis viral oncogene homolog (C-MYC),can induce pluripotency in mouse and human fibroblasts. Here,we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A],C-MYC,and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology,in the ability to differentiate into cells of the 3 embryonic layers,and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This,in turn,will allow us to better control cell-fate and apply this knowledge to cell therapy. View Publication

There is increasing evidence that many cancers,including breast cancer,contain populations of cells that display stem-cell properties. These breast cancer stem cells,by virtue of their relative resistance to radiation and cytotoxic chemotherapy,may contribute to treatment resistance and relapse. The elucidation of pathways that regulate these cells has led to the identification of potential therapeutic targets. A number of agents capable of targeting breast cancer stem cells in preclinical models are currently entering clinical trials. Assessment of the efficacy of the agents will require development of innovative clinical trial designs with appropriate biologic and clinical end points. The effective targeting of breast cancer stem cells has the potential to significantly improve outcome for women with both early-stage and advanced breast cancer. View Publication

HIV-1 infection is characterized by a progressive decline in CD4(+) T cells leading to a state of profound immunodeficiency. IL-2 therapy has been shown to improve CD4(+) counts beyond that observed with antiretroviral therapy. Recent phase III trials revealed that despite a sustained increase in CD4(+) counts,IL-2-treated patients did not experience a better clinical outcome [Abrams D,et al. (2009) N Engl J Med 361(16):1548-1559]. To explain these disappointing results,we have studied phenotypic,functional,and molecular characteristics of CD4(+) T cell populations in IL-2-treated patients. We found that the principal effect of long-term IL-2 therapy was the expansion of two distinct CD4(+)CD25(+) T cell populations (CD4(+)CD25(lo)CD127(lo)FOXP3(+) and CD4(+)CD25(hi)CD127(lo)FOXP3(hi)) that shared phenotypic markers of Treg but could be distinguished by the levels of CD25 and FOXP3 expression. IL-2-expanded CD4(+)CD25(+) T cells suppressed proliferation of effector cells in vitro and had gene expression profiles similar to those of natural regulatory CD4(+)CD25(hi)FOXP3(+) T cells (Treg) from healthy donors,an immunosuppressive T cell subset critically important for the maintenance of self-tolerance. We propose that the sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4(+) T cells had a higher relative risk of clinical progression to AIDS. View Publication

The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4,SOX2,KLF4,and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology,expression of surface antigens and pluripotency-associated transcription factors,global gene expression profiles,and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines,although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs,which corresponds to less than 1 mL of PB,was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB,with its relative efficiency and ease of harvesting,could enable the therapeutic use of patient-specific pluripotent stem cells. View Publication

Because video data are complex and are comprised of many images,mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article,we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments,hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies,recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth,three recipes were created. The first segmented the image into the colony and background,the second enhanced the image to define colonies throughout the video sequence accurately,and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes,the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared,results were virtually identical,indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition,other video bioinformatics recipes can be developed in the future for other cell processes such as migration,apoptosis,and cell adhesion. View Publication

Defining the molecular mechanisms underpinning fetal (gamma) globin gene silencing may provide strategies for reactivation of gamma-gene expression,a major therapeutic objective in patients with beta-thalassemia and sickle cell disease (SCD). We have previously demonstrated that symmetric methylation of histone H4 Arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for recruitment of the DNA methyltransferase DNMT3A to the gamma-promoter,and subsequent DNA methylation and gene silencing. Here we show in an erythroid cell line,and in primary adult erythroid progenitors that PRMT5 induces additional repressive epigenetic marks at the gamma-promoter through the assembly of a multiprotein repressor complex containing the histone modifying enzymes SUV4-20h1,casein kinase 2alpha (CK2alpha),and components of the nucleosome remodeling and histone deacetylation complex. Expression of a mutant form of PRMT5 lacking methyltransferase activity or shRNA-mediated knockdown of SUV4-20h1 resulted in loss of complex binding to the gamma-promoter,reversal of both histone and DNA repressive epigenetic marks,and increased gamma-gene expression. The repressive H4K20me3 mark induced by SUV4-20h1 is enriched on the gamma-promoter in erythroid progenitors from adult bone marrow compared with cord blood,suggesting developmental specificity. These studies define coordinated epigenetic events linked to fetal globin gene silencing,and provide potential therapeutic targets for the treatment of beta-thalassemia and SCD. View Publication

Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups,making these studies difficult. Here,we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion,the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs,especially for their homing potential. View Publication

The vast majority of pedigrees with familial Alzheimer's disease (FAD) are caused by inheritance of mutations in the PSEN1 1 gene. While genetic ablation studies have revealed a role for presenilin 1 (PS1) in embryonic neurogenesis,little information has emerged regarding the potential effects of FAD-linked PS1 variants on proliferation,self-renewal and differentiation,key events that control cell fate commitment of adult brain neural progenitors (NPCs). We used adult brain subventricular zone (SVZ)-derived NPC cultures transduced with recombinant lentivirus as a means to investigate the effects of various PS1 mutants on self-renewal and differentiation properties. We now show that viral expression of several PS1 mutants in NPCs leads to impaired self-renewal and altered differentiation toward neuronal lineage,in vitro. In line with these observations,diminished constitutive proliferation and steady-state SVZ progenitor pool size was observed in vivo in transgenic mice expressing the PS1DeltaE9 variant. Moreover,NPC cultures established from the SVZ of adult mice expressing PS1DeltaE9 exhibit reduced self-renewal capacity and premature exit toward neuronal fates. To these findings,we show that both the levels of endogenous Notch/CBF-1-transcriptional activity and transcripts encoding Notch target genes are diminished in SVZ NPCs expressing PS1DeltaE9. The deficits in self-renewal and multipotency are restored by expression of Notch1-ICD or a downstream target of the Notch pathway,Hes1. Hence,we argue that a partial reduction in PS-dependent gamma-secretase processing of the Notch,at least in part,accounts for the impairments observed in SVZ NPCs expressing the FAD-linked PS1DeltaE9 variant. View Publication

Compared with adults,pediatric mastocytosis has a relatively favorable prognosis. Interestingly,a difference was also observed in the status of c-kit mutations according to the age of onset. Although most adult patients have a D(816)V mutation in phosphotransferase domain (PTD),we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus KIT-PTD mutants were introduced into rodent Ba/F3,EML,Rat2,and human TF1 cells to investigate their biologic effect. Both ECD and PTD mutations induced constitutive receptor autophosphorylation and ligand-independent proliferation of the 3 hematopoietic cells. Unlike ECD mutants,PTD mutants enhanced cluster formation and up-regulated several mast cell-related antigens in Ba/F3 cells. PTD mutants failed to support colony formation and erythropoietin-mediated erythroid differentiation. ECD and PTD mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT,whereas AKT was only activated by ECD mutants. Consistently,AKT inhibitor suppressed ECD mutant-dependent proliferation,clonogenicity,and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EML-PTD cells,suggesting the differential role of AKT in those mutants. Overall,our study implied different pathogenesis of pediatric versus adult mastocytosis,which might explain their diverse phenotypes. View Publication

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens,including live cells,without the need for chemi-selective stains. Using a microspectrometer,near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly,compared to proteins and lipids,the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus,we could identify intensity ratios of particular protein-related bands (e.g.,757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology,proliferation,or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid,noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs. View Publication

In mouse,a subset of dendritic cells (DCs) known as CD8alpha+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However,translation into clinical protocols has been hampered by the failure to identify CD8alpha+ DCs in humans. Here,we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8alpha+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8alpha+ DCs,human DNGR-1+ BDCA3hi DCs express Necl2,CD207,BATF3,IRF8,and TLR3,but not CD11b,IRF4,TLR7,or (unlike CD8alpha+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8,but not of TLR7,and produce interleukin (IL)-12 when given innate and T cell-derived signals. Notably,DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy. View Publication