Expression data from shRFP, shATF1 in human embryonic stem cells (hESCs)
ABSTRACT: Human embryonic stem cells (hESCs) are infected by shRFP and shATF1 lentivirus for 6 days. The global gene profiles are analyzed by Affymatrix expression array. Overall design: We generated shRFP and shATF1 lentivirus and tested the titer of each virus. 10MOI of each virus were used as infection condition to infect hESCs. 6 Days infected hESCs were collected and performed microarray analysis.
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (?10,000?g) robustly produces a high-titer virus (up to 2×10(8)?TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4?°C (??1.3 days) or subjected to multiple freeze-thaw cycles (?=1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.
Project description:We used microarray analysis to study the expression differences between controls and hESCs expressing RB7LP for 3 days, and controls and hESCs expressing T121 for 3 days. RB7LP is the large pocket fragment of the retinoblastoma protein (RB) fused to GFP, in which seven phosphorylation sites for Cdk have been mutated (Angus, SP et al. Mol Cell Biol 23, 8172 (Nov, 2003). T121 is a truncated form of the SV40 Large T (LT) viral oncoprotein that inactivates the function of the three members of the retinoblastoma (RB) protein family (RB, p107, p130). We plated hESCs that had been infected with inducible lentiviruses to express either RB7LP or T121. The day after plating, one well from each virus was treated with doxycyline to induce expression of either RB7LP or T121 and after 3 days were harvested for microrarray analysis.
Project description:In this study, we developed a new purification method using chondroitin sulfate C (CSC) and protamine sulfate (PS) to concentrate lentivirus. To evaluate the efficiency of this new method, we compared it with several previously described purification protocols, including virus concentrated by ultracentrifugation (Ultra), precipitated by polyethylene glycol (PEG), and sedimented by CSC combined with polybrene (PB). After using the different methods to purify and concentrate equivalent amounts of lentivirus supernatant, the virus pellets precipitated by the different methods were resuspended using the equivalent volumes of DMEM. Subsequently, 10 ?l of each lentivirus stock carrying EGFP gene was used to transduce two types of cells, human embryonic kidney 293T (HEK293T) cells and mouse mesenchymal stem cells (mMSC). It was obvious that HEK293T and mMSC appeared much intensiver green fluorescence through virus transduction from PS method than from other methods. To quantitate the transduction efficiency of the viruses, we examined virus titer in the cells after transduction using a real-time PCR-based analysis. Accordingly, we verified that PS precipitation could generate virus with a higher titer (4.39 × 108 IU/ml) than PB (2.43 × 108 IU/ml), Ultra (1.16 × 108 IU/ml), and PEG (0.56 × 108 IU/ml) in HEK293T cells. As for HEK293T cells in mMSC, the PS method also generated virus with a higher titer (4.66 × 108 IU/ml) than the Ultra method (2.36 × 108 IU/ml), and a much higher titer than those of the other chemical-based precipitation methods using PB (4.82 × 106 IU/ml) and PEG (8.98 × 104 IU/ml). Furthermore, the HEK293T cells and mMSC transduced by PS(1X)-virus appeared to have higher cell growth ratios, respectively, than the HEK293T cells and mMSC transduced by lentivirus using the other methods. We conclude that our new method for purifying lentivirus is cost-effective, time-saving, and highly efficient, and that lentivirus purification by this means could possibly be used to transduce a variety of cells, including stem cells.
Project description:Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added to the media for 12 weeks. None of these genes are related to the germ lineage, whereas genes for neuronal cells (PAX6 and NR6A1) and endothelial cells (FLT-1 and PTF1A) were up-regulated. To induce and support the differentiation towards the germ lineage we stimulated hESCs with different concentrations of ATRA for 7 and 14 days. We observed no significant difference in gene expression on RNA level when combining all cell lines. Whereas, the overall outcome was negative, one of these cell lines demonstrated an up-regulation of DDX4 on RNA and protein level after 7 days of ATRA stimulation. In summary, our data showed that the lack of exogenous FGF2 results in up-regulation of genes crucial for neuronal and endothelial cell differentiation of hESCs, but not in the up-regulation of genes related to germ cell differentiation when cultured on hFFs. Additionally, we demonstrated that ATRA supplementation did not result in a general specific direction of hESCs towards the germ lineage.
Project description:Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.
Project description:Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage.
Project description:Differentiation of human pluripotent stem cells (hPSCs) into striatal medium spiny neurons (MSNs) promises a cell-based therapy for Huntington's disease. However, clinical-grade MSNs remain unavailable. Here, we developed a chemical recipe named XLSBA to generate clinical-grade MSNs from embryonic stem cells (ESCs). We introduced the ?-secretase inhibitor DAPT into the recipe to accelerate neural differentiation, and replaced protein components with small molecules. Using this optimized protocol we could efficiently direct regular human ESCs (hESCs) as well as clinical-grade hESCs to lateral ganglionic eminence (LGE)-like progenitors and striatal MSNs within less than half of the time than previous protocols (within 14 days and 21 days, respectively). These striatal cells expressed appropriate MSN markers and electrophysiologically acted like authentic MSNs. Upon transplantation into brains of neonatal mice or mouse model of Huntington's disease, they exhibited sufficient safety and reasonable efficacy. Therefore, this quick and highly efficient derivation of MSNs offers unprecedented access to clinical application.
Project description:Overexpression of NEUROG2 and NEUROG1 (NEUROG2/1) in human embryonic stem cells (hESCs) rapidly produces functional networks of excitatory and inhibitory neurons. To facilitate the use of this efficient inducible human neuron model in neuroscience research, we generated hESCs with doxycycline-inducible NEUROG2/1 via lentivirus and a tdTomato fluorescent reporter knock-in at the MAP2 locus using the CRISPR nuclease Cas9. Upon doxycycline-driven induction of NEUROG2/1, these hESCs differentiate within days into cells that are uniformly MAP2 immunoreactive and tdTomato fluorescent.
Project description:Human embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.
Project description:BACKGROUND:Mesenchymal stem/stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are attractive for their hematopoietic-supporting or potential therapeutic effects. However, procedures for high-effective and scalable generation of MSCs from hESCs within 2 weeks are still unestablished, which also hinder the development and mechanism study of mesengenesis. METHODS:In this study, we aimed to establish a strategy for programming hESC differentiation into MSCs by practicing small-scale chemical compound screening. Then, we used flow cytometry, multi-lineage differentiation, and karyotype analyses to investigate the biological phenotypes of the derived hESC-MSCs. Also, to explore whether the derived cells had hematopoietic-supporting ability in vitro, we carried out the cobblestone formation and megakaryocytic differentiation experiments. To further evaluate the function of hESC-MSCs in vivo, we transplanted the cells into a mouse model with hind limb ischemia. RESULTS:By simultaneous treatments with a JAK/STAT antagonist and a DNA methylation inhibitor, the efficiency of generating hESCs into CD73+ hESC-MPCs could reach 60% within 7 days. The derived cells further matured into hESC-MSCs, with comparable characteristics to those of adult MSCs in terms of surface markers, normal karyotype, and the potential for adipogenic, osteogenic, and chondrogenic differentiation. Functionally, hESC-MSCs had hematopoietic-supporting effects in vitro and could notably relieve symptoms of hind limb ischemia. CONCLUSIONS:In the study, we established a high-efficient procedure for large-scale generation of MSCs from hESCs, which would be of great help for genesis and mechanism studies of MSCs. Meanwhile, the derived cells provide an alternative for translational clinical research.