搜索结果: 'EasySep Direct'

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine,levels of which are modulated by stress,depression and clinical antidepressants. However,studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors,including stem cells,from the hippocampus of adult mice. Mechanistically,we provide evidence that beta(3)-adrenergic receptors,which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ),mediate this norepinephrine-dependent activation. Moreover,intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly,systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally,using a novel ex vivo slice-sphere" assay that maintains an intact neurogenic niche� View Publication

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however,the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects,with a median of 14 individual epitopic regions targeted per person (range,2 to 42),and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median,4,245) among all study participants. However,the number of epitopic regions targeted,the protein subunits recognized,and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals,with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid,sensitive,specific,and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response,even if a comprehensive pan-genome screening approach is applied. View Publication

Bone marrow-derived endothelial precursor cells incorporate into neovasculature and have been successfully used as vehicles for gene delivery to brain tumors. To determine whether systemically administered Sca1+ bone marrow cells labeled with superparamagnetic iron oxide nanoparticles can be detected by in vivo magnetic resonance imaging in a mouse brain tumor model,mouse Sca1+ cells were labeled in vitro with ferumoxides-poly-L-lysine complexes. Labeled or control cells were administered intravenously to glioma-bearing severe combined immunodeficient (SCID) mice. Magnetic resonance imaging (MRI) was performed during tumor growth. Mice that received labeled cells demonstrated hypointense regions within the tumor that evolved over time and developed a continuous dark hypointense ring at a consistent time point. This effect was not cleared by administration of a gadolinium contrast agent. Histology showed iron-labeled cells around the tumor rim in labeled mice,which expressed CD31 and von Willebrand factor,indicating the transplanted cells detected in the tumor have differentiated into endothelial-like cells. These results demonstrate that MRI can detect the incorporation of magnetically labeled bone marrow-derived precursor cells into tumor vasculature as part of ongoing angiogenesis and neovascularization. This technique can be used to directly identify neovasculature in vivo and to facilitate gene therapy by noninvasively monitoring these cells as gene delivery vectors. View Publication

TEL-TRKC is a fusion gene generated by chromosomal translocation and encodes an activated tyrosine kinase. Uniquely,it is found in both solid tumors and leukemia. However,a single exon difference (in TEL) in TEL-TRKC fusions is associated with the two sets of cancer phenotypes. We expressed the two TEL-TRKC variants in vivo by using the 3' regulatory element of SCL that is selectively active in a subset of mesodermal cell lineages,including endothelial and hematopoietic stem cells and progenitors. The leukemia form of TEL-TRKC (-exon 5 of TEL) enhanced hematopoietic stem cell renewal and initiated leukemia. In contrast,the TEL-TRKC solid tumor variant (+ TEL exon 5) elicited an embryonic lethal phenotype with impairment of both angiogenesis and hematopoiesis indicative of an effect at the level of the hemangioblasts. The ability of TEL-TRKC to repress expression of Flk1,a critical regulator of early endothelial and hematopoietic cells,depended on TEL exon 5. These data indicate that related oncogenic fusion proteins similarly expressed in a hierarchy of early stem cells can have selective,cell type-specific developmental impacts. View Publication

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication

The pluripotent property of hESCs makes them attractive for treatment of degenerative diseases such as diabetes. We have developed a stage-wise directed differentiation protocol to produce alginate-encapsulated islet-like cells derived from hESCs,which can be directly implanted for diabetes therapy. The advantage of alginate encapsulation lies in its capability to immunoisolate,along with the added possibility of scalable culture. We have evaluated the possibility of encapsulating hESCs at different stages of differentiation. Encapsulation of predifferentiated cells resulted in insufficient cellular yield and differentiation. On the other hand,encapsulation of undifferentiated hESCs followed by differentiation induction upon encapsulation,resulted in the highest viability and differentiation. More striking was that alginate encapsulation resulted in a much stronger differentiation compared to parallel 2D cultures,resulting in 20-fold increase in c-peptide protein synthesis. To elucidate the mechanism contributing to encapsulation-mediated enhancement in hESC maturation,investigation of the signaling pathways revealed interesting insight. While the phospho-protein levels of all the tested signaling molecules were lower under encapsulation,the ratio of pSMAD/pAKT was significantly higher,indicating a more efficient signal transduction under encapsulation. These results clearly demonstrate that alginate encapsulation of hESCs and differentiation to islet-cells types provides a potentially translatable treatment option for type1 diabetes. View Publication

Human genome engineering has been transformed by the introduction of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system found in most bacteria and archaea. Type II CRISPR/Cas systems have been engineered to induce RNA-guided genome editing in human cells,where small RNAs function together with Cas9 nucleases for sequence-specific cleavage of target sequences. Here we describe the protocol for Cas9-mediated human genome engineering,including construct building and transfection methods necessary for delivering Cas9 and guide RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells. Following genome editing,we also describe methods to assess genome editing efficiency using next-generation sequencing and isolate monoclonal hiPSCs with the desired modifications for downstream applications. View Publication

Differentiation of human stem cells to hepatocytes is crucial for industrial applications as well as to develop new therapeutic strategies for liver disease. The protocol described here,using sequentially growth factors known to play a role in liver embryonic development,efficiently differentiates human embryonic stem cells (hESC) as well as human-induced pluripotent stem cells (hiPSC) to hepatocytes by directing them through defined embryonic intermediates,namely,mesendoderm/definitive endoderm and hepatoblast and hepatocyte phenotype. After 28 days,the final differentiated progeny is a mixture of cells,comprising cells with characteristics of hepatoblasts and a smaller cell fraction with morphological and phenotypical features of mature hepatocytes. An extensive functional characterization of the stem cell progeny should be used to confirm that differentiated cells display functional characteristics of mature hepatocytes including albumin secretion,glycogen storage,and several detoxifying functions such as urea production,bilirubin conjugation,glutathione S-transferase activity,cytochrome activity and drug transporter activity. View Publication

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson's disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development,A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here,we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons,which mimics embryonic DA neuron development. In our protocol,we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method,and then convert the FP cells to A9 DA neurons,which could be maintained in vitro for several months. This efficient,repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients,in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD. View Publication

Based on knowledge of early embryo development,where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling,most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and,when considering clinical applications,have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function,for understanding disease processes,for use in drug discovery,and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here,we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways,and in animal-component-free conditions,replacing the frequently used Noggin and SB431542. We also show by transcript analysis,both at the population level and for the first time at the single-cell level,that differentiated cells express genes characteristic for the development of ANE,in particular for the development of the future forebrain. View Publication

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential,however,depends on the availability of culture methods that are robust,scalable,and use chemically defined materials. Despite significant advances in hiPSC technologies,the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts,such as Matrigel,which raises safety concerns over the use of these products. In this work,we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined,xeno-free synthetic peptide substrate,i.e. Corning Synthemax(®) Surface. We demonstrated that the Synthemax Surface supports the attachment,spreading,and proliferation of hiPSCs,as well as hiPSCs' lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α(v)β(5) integrins to mediate attachment to the substrate,whereas multiple integrins are involved in cell attachment to Matrigel. Finally,hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials. View Publication

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases,including schizophrenia,autism,and epilepsy. Here,we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles,neurotransmitter phenotypes,and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology. View Publication