搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. View Publication

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. View Publication

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general,but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies,however,is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is,however,limited and thereby further optimization in terms of time,efficiency,and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts,at least in part,in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1,Inhba and Grem1. Hence,we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. View Publication

Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29. days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5. days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2?? was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2?? was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells,but its downregulation was much slower in HEK293 cells. Additionally,two ER-resident E3 ubiquitin ligases,gp78 and Hrd1,were both upregulated in H9 cells following 5. days of exposure to RA. Moreover,the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells,and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29. days,GRP78/Bip,XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA. ?? 2011 Elsevier Inc. View Publication

The chemical approach to controlling stem cell fates is emerging as a powerful tool,holding great promise in tissue engineering and regenerative medicine. Various small molecules have been demonstrated capable of modulating stem cell differentiation. In this paper,we studied the effects of 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAR),an activator of AMP-activated protein kinase (AMPK),on mesenchymal stem cells (MSCs). AICAR at high concentrations (1.0-2.0 mM) significantly inhibited proliferation of both human amnion-derived MSCs (hAMSCs) and rabbit bone marrow-derived MSCs (BM-MSCs). Most importantly,AICAR efficiently promoted the osteogenic differentiation of hAMSCs and BM-MSCs in both growth medium and osteogenic medium. However,Metformin,another AMPK activator,showed no such effects. Meanwhile,AICAR significantly inhibited adipogenic differentiation of hAMSCs and BM-MSCs. Our data suggests that AICAR represents a potent molecule,which can be applied in bone tissue regeneration. View Publication

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting� View Publication

IL-17-producing CD4+ T helper cells (TH17) have been extensively investigated in mouse models of autoimmunity. However,the requirements for differentiation and the properties of pathogen-induced human TH17 cells remain poorly defined. Using an approach that combines the in vitro priming of naive T cells with the ex vivo analysis of memory T cells,we describe here two types of human TH17 cells with distinct effector function and differentiation requirements. Candida albicans-specific TH17 cells produced IL-17 and IFN-γ,but no IL-10,whereas Staphylococcus aureus-specific TH17 cells produced IL-17 and could produce IL-10 upon restimulation. IL-6,IL-23 and IL-1β contributed to TH17 differentiation induced by both pathogens,but IL-1β was essential in C. albicans-induced TH17 differentiation to counteract the inhibitory activity of IL-12 and to prime IL-17/IFN-γ double-producing cells. In addition,IL-1β inhibited IL-10 production in differentiating and in memory TH17 cells,whereas blockade of IL-1β in vivo led to increased IL-10 production by memory TH17 cells. We also show that,after restimulation,TH17 cells transiently downregulated IL-17 production through a mechanism that involved IL-2-induced activation of STAT5 and decreased expression of ROR-γt. Taken together these findings demonstrate that by eliciting different cytokines C. albicans and S. aureus prime TH17 cells that produce either IFN-γ or IL-10,and identify IL-1β and IL-2 as pro- and anti-inflammatory regulators of TH17 cells both at priming and in the effector phase. View Publication

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here,we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition,which offer an efficient formation of aggregates. To specify the neuroectodermal specification,these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes. View Publication

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K,which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 $\mu$mol/L (MCF7 cells) and 0.17 $\mu$mol/L (SW620 cells),and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 $\mu$mol/L (MCF7 cells) and more than 10 $\mu$mol/L (SW620 cells). Five-day exposure to 1 $\mu$mol/L KU-0060648 inhibited cell proliferation by more than 95{\%} in MCF7 cells but only by 55{\%} in SW620 cells. In clonogenic survival assays,KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells,but not in DNA-PKcs-deficient cells,thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts,concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold,without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors. View Publication

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics,we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo,LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3-/- CD4+ T cells,solidifying that LAG-3 controls these processes. Lag3-/- CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation,enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally,enhancement of STAT5 activation,as a result of LAG-3 deficiency,contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. View Publication

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures,where long single-stranded DNA is folded into a compact nanostructure,present an attractive approach to package genes; however,effective delivery of genetic material into cell nuclei has remained a critical challenge. Here,we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore,our study validates virus-like particles as an efficient method of DNA nanostructure delivery,opening the possibility of delivering nanostructures in vivo to specific cell types. Together,these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates,exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions,such as biosensing,into cell nuclei. View Publication

Adipose tissue dysfunction is strongly linked to the development of chronic inflammation and cardiometabolic disorders in aging. While much attention has been given to the role of resident adipose tissue immune cells in the disruption of homeostasis in obesity,age-specific effects remain understudied. Here,we identified and characterized a population of ?? T cells,which show unique age-dependent accumulation in the visceral adipose tissue (VAT) of both mice and humans. Diet-induced obesity likewise increased ?? T cell numbers; however,the effect was greater in the aged where the increase was independent of fat mass. ?? T cells in VAT express a tissue-resident memory T cell phenotype (CD44hiCD62LlowCD69+) and are predominantly IL-17A-producing cells. Transcriptome analyses of immunomagnetically purified ?? T cells identified significant age-associated differences in expression of genes related to inflammation,immune cell composition,and adipocyte differentiation,suggesting age-dependent qualitative changes in addition to the quantitative increase. Genetic deficiency of ?? T cells in old age improved the metabolic phenotype,characterized by increased respiratory exchange ratio,and lowered levels of IL-6 both systemically and locally in VAT. Decreased IL-6 was predominantly due to reduced production by non-immune stromal cells,primarily preadipocytes,and adipose-derived stem cells. Collectively,these findings suggest that an age-dependent increase of tissue-resident ?? T cells in VAT contributes to local and systemic chronic inflammation and metabolic dysfunction in aging. View Publication