技术资料

Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors,but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular,valproic acid (VPA),an HDAC inhibitor,improves reprogramming efficiency by more than 100-fold,using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc. View Publication

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs,or treatment with recombinant BMP4,with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin,a BMP antagonist,demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore,BECs may provide a feedback mechanism to control the proliferation of NSPCs. View Publication

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation,a common approach to mimic some of the spatial and temporal aspects of in vivo development,are affected by three factors: input hESC composition,input hESC colony size,and EB size. Using a microcontact printing approach,size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently,size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover,upon forming EBs from these MP-hESCs,we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs,higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely,neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size,typical in most differentiation strategies,produces subsets of appropriate conditions for differentiation into specific cell types. Moreover,our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories. View Publication

Outcomes of 159 young patients with inherited metabolic disorders (IMDs) undergoing transplantation with partially HLA-mismatched unrelated donor umbilical cord blood were studied to investigate the impact of graft and patient characteristics on engraftment,overall survival (OS),and graft-versus-host disease (GVHD). Patients received myeloablative chemotherapy (busulfan,cyclophosphamide,ATG) and cyclosporine-based GVHD prophylaxis. Infused cell doses were high (7.57 x 10(7)/kg) because of the patients' young age (median,1.5 years) and small size (median,12 kg). Median follow-up was 4.2 years (range,1-11 years). The cumulative incidences of neutrophil and platelet engraftment were 87.1% (95% confidence interval [CI],81.8%-92.4%) and 71.0% (95% CI,63.7%-78.3%). A total of 97% achieved high (textgreater 90%) donor chimerism. Serum enzyme normalized in 97% of patients with diseases for which testings exist. Grade III/IV acute GVHD occurred in 10.3% (95% CI,5.4%-15.2%) of patients. Extensive chronic GVHD occurred in 10.8% (95% CI,5.7%-15.9%) of patients by 1 year. OS at 1 and 5 years was 71.8% (95% CI,64.7%-78.9%) and 58.2% (95% CI,49.7%-66.6%) in all patients and 84.5% (95% CI,77.0%-92.0%) and 75.7% (95% CI,66.1%-85.3%) in patients with high (80-100) performance score. In multivariate analysis,favorable factors for OS were high pretransplantation performance status,matched donor/recipient ethnicity,and higher infused colony forming units. View Publication

Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90,resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study,we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG,as well as enhanced 17-AAG-mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably,treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition,cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG. View Publication

The cancer stem cell hypothesis proposes that cancers arise in stem/progenitor cells through disregulation of self-renewal pathways generating tumors,which are driven by a component of 'tumor-initiating cells' retaining stem cell properties. The HER2 gene is amplified in 20-30% of human breast cancers and has been implicated in mammary tumorigenesis as well as in mediating aggressive tumor growth and metastasis. We demonstrate that HER2 overexpression drives mammary carcinogenesis,tumor growth and invasion through its effects on normal and malignant mammary stem cells. HER2 overexpression in normal mammary epithelial cells (NMEC) increases the proportion of stem/progenitor cells as demonstrated by in vitro mammosphere assays and the expression of stem cell marker aldehyde dehydrogenase (ALDH) as well as by generation of hyperplastic lesions in humanized fat pads of NOD (nucleotide-binding oligomerization domain)/SCID (severe combined immunodeficient) mice. Overexpression of HER2 in a series of breast carcinoma cell lines increases the ALDH-expressing 'cancer stem cell' population which displays increased expression of stem cell regulatory genes,increased invasion in vitro and increased tumorigenesis in NOD/SCID mice. The effects of HER2 overexpression on breast cancer stem cells are blocked by trastuzumab in sensitive,but not resistant,cell lines,an effect mediated by the PI3-kinase/Akt pathway. These studies provide support for the cancer stem cell hypothesis by suggesting that the effects of HER2 amplification on carcinogenesis,tumorigenesis and invasion may be due to its effects on normal and malignant mammary stem/progenitor cells. Furthermore,the clinical efficacy of trastuzumab may relate to its ability to target the cancer stem cell population in HER2-amplified tumors. View Publication

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process,but the molecular mechanisms involved,particularly in the human mammary gland,are poorly understood. To address this issue,we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified,and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together,these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation. View Publication

Murine embryonic stem (mES) cells are self-renewing pluripotent cells that bear the capacity to differentiate into ectoderm-,endoderm-,and mesoderm-derived tissues. In suspension culture,embryonic stem (ES) cells grow into spherical embryoid bodies (EBs) and are useful for the study of specific gene products in the development and function of various tissue types. Osteoclasts are hematopoietic stem cell-derived cells that participate in bone turnover by secreting resorptive molecules such as hydrochloric acid and acidic proteases,which degrade the bone extracellular matrix. Aberrant osteoclast function leads to dysplastic,erosive,and sclerosing bone diseases. Previous studies have reported the derivation of osteoclasts from mES cells; however,most of these protocols require coculture with stromal cell lines. We describe two simplified,novel methods of stromal cell-independent ES cell-derived osteoclast development. View Publication

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However,it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous,endogenously HIV-1-infected CD4+ T cells. Here,we stimulate primary CD4+ T cells,purified ex vivo from HIV-1-infected viremic patients,with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that,subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules,HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells,while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However,the degree of NK cell cytolytic activity against autologous,endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively,our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells. View Publication

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors,cytokines,and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover,ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study,we evaluated the effects of ascorbic acid on the proliferation,differentiation,extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks,following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover,the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration. View Publication

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways,as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study,we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation,neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs,whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors,of which type I IFNs were one of the active components,played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types,MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore,signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs,with MyD88 playing a larger role than TRIF. In sum,in our experimental systems,TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation. View Publication

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory,CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous,consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach,we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+),yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts,both in vitro and in vivo. These data,combined with those from our previous work,reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) textgreater CD133(+)ALDH(-) textgreater CD133(-)ALDH(-). ALDH,expressed along CD133,can more specifically characterize the tumorigenic liver CSC population. View Publication