技术资料

We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct,allowing its regulated expression (and,at the same time,deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes,miRNA-based RNAi (including two shRNA constructs at once),and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture,efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny,and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo. View Publication

The developmental potential of human ES cells makes them an important tool in developmental,pharmacological,and clinical research. For human ES cell technology to be fully exploited,however,culture efficiency must be improved,large-scale culture enabled,and safety ensured. Traditional human ES cell culture systems have relied on serum products and mouse feeder layers,which limit the scale,present biological variability,and expose the cells to potential contaminants. Defined,feeder-independent culture systems improve the safety and efficiency of ES cell technology,enabling translational research. The protocols herein are designed with the standard research laboratory in mind. They contain recipes for the formulation of mTeSR (a defined medium for human ES cell culture) and detailed protocols for the culture,transfer,and passage of cells grown in these feeder-independent conditions. They provide a basis for routine feeder-independent culture,and a starting point for additional optimization of culture conditions. View Publication

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4,SOX2,and either cMYC and KLF4 or NANOG and LIN28. Little is known,however,about the mechanisms by which reprogramming occurs,which is in part limited by the low efficiency of conversion. To this end,we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs),demonstrated by gene expression profiles,DNA methylation status,and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts,we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system,we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells,we obtained secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming." View Publication

Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous secondary" somatic cells� View Publication

Adipose tissue development is associated with neovascularization,which might be exploited therapeutically. We investigated the neovasculogenesis antigenic profile and kinetics in adipose tissue-derived stromal cells (ADSCs) to understand the potential of ADSCs to generate new vessels. Murine and human visceral adipose tissues were processed with collagenase to obtain ADSCs from the stromal vascular fraction. Freshly isolated murine and human ADSCs featured the expression of early markers of endothelial differentiation [uptake of DiI-labeled acetylated LDL,CD133,CD34,kinase insert domain receptor (KDR)],but not markers for more mature endothelial cells (CD31 and von Willebrand factor). In methylcellulose medium,multilocular cells positive for Oil Red O staining appeared after 6 days. After 10 days,clusters of ADSCs spontaneously formed branched tubelike structures,which were strongly positive for CD34 and CD31,while losing their ability to undergo adipocyte differentiation. In Matrigel,in the presence of endothelial growth factors ADSCs formed branched tubelike structures. By clonal assays in methylcellulose we also determined the frequency of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming units from ADSCs,compared with bone marrow-derived stromal cells (BMSCs) used as a positive control. After 4-14 days,BMSCs formed 8 +/- 3 BFU-E and 40 +/- 10 CFU-GM,while ADSCs never produced colonies of myeloid progenitors. The developing adipose tissue has neovasculogenic potential,based on the recruitment of local rather than circulating progenitors. Adipose tissue might therefore be a viable autonomous source of cells for postnatal neovascularization. View Publication

OBJECTIVE: The T-cell receptor zeta (TCR zeta)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCR zeta-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCR zeta(dim) T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCR zeta(dim) T cells in coronary artery disease. METHODS AND RESULTS: We examined the expression of TCR zeta-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4(+) TCR zeta(dim) T cells were found in patients with acute coronary syndromes (ACS,n=66; median 5.3%,interquartile 2.6 to 9.1% of total CD4(+) T cells; Ptextless0.0001) compared to chronic stable angina (CSA,n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein,and it persisted after the acute event. Moreover,TCR zeta(dim) cells were also more represented within CD8(+) T cell,NK,and CD4(+)CD28(null) T cell subsets in ACS compared to CSA and controls. Finally,CD4(+) and CD8(+) TCR zeta(dim) T cells isolated from ACS displayed an enhanced transendothelial migratory capacity. CONCLUSIONS: TCR zeta(dim) T cells,an effector T-cell subset with transendothelial migratory ability,are increased in ACS,and may be implicated in coronary instability. View Publication

Recent evidence suggests that blockade of aberrant Hedgehog signaling can be exploited as a therapeutic strategy for pancreatic cancer. Our previous studies using the prototype Hedgehog small-molecule antagonist cyclopamine had shown the striking inhibition of systemic metastases on Hedgehog blockade in spontaneously metastatic orthotopic xenograft models. Cyclopamine is a natural compound with suboptimal pharmacokinetics,which impedes clinical translation. In the present study,a novel,orally bioavailable small-molecule Hedgehog inhibitor,IPI-269609,was tested using in vitro and in vivo model systems. In vitro treatment of pancreatic cancer cell lines with IPI-269609 resembled effects observed using cyclopamine (i.e.,Gli-responsive reporter knockdown,down-regulation of the Hedgehog target genes Gli1 and Ptch,as well as abrogation of cell migration and colony formation in soft agar). Single-agent IPI-269609 profoundly inhibited systemic metastases in orthotopic xenografts established from human pancreatic cancer cell lines,although Hedgehog blockade had minimal effect on primary tumor volume. The only discernible phenotype observed within the treated primary tumor was a significant reduction in the population of aldehyde dehydrogenase-bright cells,which we have previously identified as a clonogenic tumor-initiating population in pancreatic cancer. Selective ex vivo depletion of aldehyde dehydrogenase-bright cells with IPI-269609 was accompanied by significant reduction in tumor engraftment rates in athymic mice. Pharmacologic blockade of aberrant Hedgehog signaling might prove to be an effective therapeutic strategy for inhibition of systemic metastases in pancreatic cancer,likely through targeting subsets of cancer cells with tumor-initiating (cancer stem cell") properties." View Publication

With their unique ability to differentiate into all cell types,embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation,we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative,hold the greatest differentiation potential,and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation,we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates,we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system,similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation. View Publication

Epithelial-mesenchymal transition (EMT) plays important roles in various physiological and pathological processes,and is regulated by signaling pathways mediated by cytokines,including transforming growth factor beta (TGFbeta). Embryonic endothelial cells also undergo differentiation into mesenchymal cells during heart valve formation and aortic maturation. However,the molecular mechanisms that regulate such endothelial-mesenchymal transition (EndMT) remain to be elucidated. Here we show that TGFbeta plays important roles during mural differentiation of mouse embryonic stem cell-derived endothelial cells (MESECs). TGFbeta2 induced the differentiation of MESECs into mural cells,with a decrease in the expression of the endothelial marker claudin 5,and an increase in expression of the mural markers smooth muscle alpha-actin,SM22alpha and calponin,whereas a TGFbeta type I receptor kinase inhibitor inhibited EndMT. Among the transcription factors involved in EMT,Snail was induced by TGFbeta2 in MESECs. Tetracycline-regulated expression of Snail induced the differentiation of MESECs into mural cells,whereas knockdown of Snail expression abrogated TGFbeta2-induced mural differentiation of MESECs. These results indicate that Snail mediates the actions of endogenous TGFbeta signals that induce EndMT. View Publication

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors,where it initiates critical cell survival and proliferation signals after stimulation by its ligand,thrombopoietin (TPO). As a result,a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore,they show very low-level expression of Mpl on platelets and megakaryocytes,and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However,impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels,which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis. View Publication