技术资料

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface,and their removal from CM impairs proliferation. Finally,we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography,immunoblotting,and mass spectrometry-based proteomic analysis,we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture. 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

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

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

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

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

Spinal muscular atrophy (SMA),a motor neuron disease (MND) and one of the most common genetic causes of infant mortality,currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study,we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function,increased life span,and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern,including changes in RNA metabolism proteins,cell cycle proteins,and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype,indicating that transplantation of NSCs may be a possible treatment for SMA. View Publication

MSCs are known to have an extensive proliferative potential and ability to differentiate in various cell types. Osteoblastic differentiation from mesenchymal progenitor cells is an important step of bone formation,though the pattern of gene expression during differentiation is not yet well understood. Here,to investigate the possibility to obtain a model for in vitro bone differentiation using mesenchymal stem cells (hMSCs) from human subjects non-invasively,we developed a method to obtain hMSCs-like cells from peripheral blood by a two step method that included an enrichment of mononuclear cells followed by depletion of unwanted cells. Using these cells,we analyzed the expression of transcription factor genes (runt-related transcription factor 2 (RUNX2) and osterix (SP7)) and bone related genes (osteopontin (SPP1),osteonectin (SPARC) and collagen,type I,alpha 1 (COLIA1)) during osteoblastic differentiation. Our results demonstrated that hMSCs can be obtained from peripheral blood and that they are able to generate CFU-F and to differentiate in osteoblast and adipocyte; in this study,we also identified a possible gene expression timing during osteoblastic differentiation that provided a powerful tool to study bone physiology. View Publication

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin,causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs,when cotransfected with recombinatory donor DNA fragments,can promote single base-pair modification at the start of the second intron of the beta-globin gene,the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose,sequence,cell-cycle stage,and the presence of a homologous donor DNA molecule. Enhanced recombination,with frequencies up to 0.4%,was observed with use of the lysomotropic agent chloroquine. Finally,we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells,including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable,site-specific modification of disease-related genes in human cells. View Publication

Thrombocytopenia is a critical problem that occurs in many hematologic diseases,as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization,availability,and expense. Thus,the development of safe,small,molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein,we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)),increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly,the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma,but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively,these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients. View Publication

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However,successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models,the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary,we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury,KDR,CD31 and Lmo2 gene expression,while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development,consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs,and although simplified,results in an eightfold increase in cell yield. View Publication

Given their self-renewing and pluripotent capabilities,human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However,the host immune response against transplanted hESCs is not well characterized. In fact,controversy remains as to whether hESCs have immune-privileged properties. To address this issue,we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death,suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses,resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover,we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally,we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together,these data suggest that hESCs are immunogenic,trigger both cellular and humoral-mediated pathways,and,as a result,are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches. View Publication