技术资料

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is a crucial step in cell therapy. In the present study,we demonstrate that functional CD34(+) progenitor cells can be efficiently produced from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher proportion of CD34(+) cells (∼ 20%) could be derived from hPSCs by inhibition of mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling and activation of bone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34(+) progenitor cells further developed to endothelial and smooth muscle cells with functionality. Moreover,they contributed directly to neovasculogenesis in ischemic mouse hind limbs,thereby resulting in improved blood perfusion and limb salvage. Our results suggest that combined modulation of signaling pathways may be an efficient means of differentiating hPSCs into functional CD34(+) progenitor cells. View Publication

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However,G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors,precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently,new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice,we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF,implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment,genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42),and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View Publication

Chemical compounds have emerged as powerful tools for modulating ESC functions and deriving induced pluripotent stem cells (iPSCs),but documentation of compound-induced efficient directed differentiation in human ESCs (hESCs) and human iPSC (hiPSCs) is limited. By screening a collection of chemical compounds,we identified compound C (also denoted as dorsomorphin),a protein kinase inhibitor,as a potent regulator of hESC and hiPSC fate decisions. Compound C suppresses mesoderm,endoderm,and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in both hESCs and hiPSCs,88.7% and 70.4%,respectively. Interestingly,compound C is ineffective in inducing neural conversion in mouse ESCs (mESCs). Large-scale kinase assay revealed that compound C targets at least seven transforming growth factor beta (TGF-β) superfamily receptors,including both type I and type II receptors,and thereby blocks both the Activin and bone morphogenesis protein (BMP) signaling pathways in hESCs. Dual inhibition of Activin and BMP signaling accounts for the effects of compound C on hESC differentiation and neural conversion. We also identified muscle segment homeobox gene 2 (MSX2) as a downstream target gene of compound C and a key signaling intermediate of the BMP pathway in hESCs. Our findings provide a single-step cost-effective method for efficient derivation of neural progenitor cells in adherent culture from human pluripotent stem cells. Therefore,it will be uniquely suitable for the production of neural progenitor cells in large scale and should facilitate the use of stem cells in drug screening and regenerative medicine and study of early human neural development. View Publication

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells,including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate,as well as reversing those decisions during induced pluripotency (iPS),have focused largely on transcriptomic controls. Here,we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore,p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However,expression of constitutively active p70 S6K,but not wild-type p70 S6K,induces differentiation. Additionally,hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation. View Publication

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years,significant developments in stem cell research have been applied to MNDs,particularly regarding neuroprotection and cell replacement. However,a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types,including motor neurons that could be used for MND therapies. Recently,it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons,since they share ES characteristics,self-renewal,and the potential to differentiate into any somatic cell type. In this review,we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons,and possible clinical applications. View Publication

Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. We investigated the ability of two commercially available electroporation systems,the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However,the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency,producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally,we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. Our results indicate that these electroporation methods provide a reliable,efficient,and high-throughput approach to the genetic manipulation of hESC. View Publication

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models,genomic health analyses,and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small,clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology� View Publication

Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study,we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters,including the number of cells in a colony,colony area and shape,intensity of nuclear staining,and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60),as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries,and identified 17 that promoted differentiation,as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug,pinacidil,which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2,ROCK,MNK1,RSK1,and MSK1 kinases may contribute to the survival of hESCs. View Publication

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511,a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component,human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages),after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps,hES cells spread out in a monolayer,maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes. View Publication

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost,robust,scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined,xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally,PAS were scaled up to large culture-vessel formats. Synthetic,xeno-free,scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies. View Publication

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

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2,NANOG,and OCT-4,the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here,we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency,prevents cell proliferation,and enhances mesoderm and endoderm activities in hESCs. At the molecular level,mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growth-inhibitory genes,as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration. View Publication