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The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However,the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study,we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow,in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells,NK progenitors,and NKG2A-negative NK cells),and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore,we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally,we show that acetylated histones are associated with the CpG-rich region in NKG2A positive,but not negative,cell lines,and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription,but cotreatment with both drugs resulted in a significantly greater induction,suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells. View Publication

A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However,standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium,imposing a significant obstacle to clinical translation. Here,we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions,we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently,transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal,normal karyotype and pluripotency,as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly,we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications. textcopyright 2013 Elsevier Ltd. View Publication

T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However,whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition,GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue,as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast,under non-competitive conditions,GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas,the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection,GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly,we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR-/- counterparts. Thus,GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation. View Publication

Monocyte cytokines (ie,monokines) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma),which is critical for monocyte clearance of infectious pathogens and tumor surveillance. Human CD56bright NK cells produce far more IFN-gamma in response to monokines than do CD56dim NK cells. The kinases and phosphatases involved in regulating IFN-gamma production by monokine-activated NK cells are not clearly identified. SHIP1 is a 5' inositol phosphatase that dephosphorylates the phosphatidylinositol-3 kinase (PI-3K) product PI3,4,5P3. Here,we show that constitutive expression of SHIP1 is distinctly lower in CD56bright NK cells compared with CD56dim NK cells,suggesting it could be an important negative regulator of IFN-gamma production in monokine-activated NK cells. Indeed,overexpression of SHIP1 in CD56bright NK cells followed by monokine activation substantially lowered IFN-gamma production. This effect was not seen when NK cells were infected with a SHIP1 mutant containing an inactive catalytic domain. Finally,NK cells in SHIP1-/- mice produced more IFN-gamma in response to monokines in vivo than did NK cells from wild-type mice. Collectively,these results demonstrate that SHIP1 negatively regulates monokine-induced NK cell IFN-gamma production in vitro and in vivo and provide the first molecular explanation for an important functional distinction observed between CD56bright and CD56dim human NK subsets. View Publication

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1$$ (HIF-1$$) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1$$ in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study,we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1$$ and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1$$. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats,DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone,leading to bone regeneration. Collectively,the results indicate that DMOG,via activation of the PI3K/Akt pathway,promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration. View Publication

Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use,in either the autologous or allogeneic setting,cEPCs should likely be expanded from CB kept frozen in CB banks. In this study,we compared the expansion,functional features,senescence pattern over culture,and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB,while CB units were matched in terms of initial volume,nucleated and CD34(+) cell number. Moreover,the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established,the proliferation,migration,tube formation,and acetylated-LDL uptake potentials were similar in both groups. In addition,cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo,in a hind limb ischemia murine model,cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus,cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However,the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use. View Publication

Current EC differentiation protocols are inefficient,and the phenotypes of the differentiated ECs are only briefly stable,which significantly inhibits their utility for basic science research. Here,a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol,up to 45% of the differentiated hiPSCs assumed an EC phenotype,and after purification,greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks invitro. Gene and protein expression levels of CD31,CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively,these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and,furthermore,the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks invitro. ?? 2014 Elsevier Ltd. View Publication

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However,intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover,evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation,with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive,its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5(+) (up to 98%) populations. These Nkx2.5(+) populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine. View Publication

Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis,which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However,little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1,whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs,we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type. View Publication

A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8 effector T cells derived from CD45RA(-) CD62L(+) central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here,we describe a clinical scale,closed system,immunomagnetic selection method to isolate CD8(+) T(CM) from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14(+),CD45RA(+),and CD4(+) cells from PBMC,and then to positively select CD62L(+) cells. The average purity and yield of CD8(+) CD45RA(-) CD62L TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC),respectively. These CD8(+) T(CM) are responsive to anti-CD3/CD28 bead stimulation,and can be efficiently transduced with CAR encoding lentiviral vectors,and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3-6 weeks. The resulting CD8(+) T(CM)-derived effectors are polyclonal,retain expression of CD62L and CD28,exhibit CAR-redirected antitumor effector function,and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified T(CM) cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19(+) B-cell malignancies,and 3 clinical cell products expressing a CD19-specific CAR for IND 14645 have already been successfully generated from lymphoma patients using this manufacturing platform. View Publication

Glioblastomas (GBMs) are highly aggressive,invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these,cells endowed with stem properties,tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells,termed cancer stem-like cells,have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs),a family of membrane receptors,play a prominent role in cell signaling,cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here,we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs),U-87 MG cells,human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated,138 were retained for comparative studies between the different cell types. At the transcriptomic level,eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets. View Publication