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

The neurosphere assay (NSA) is one of the most frequently used methods to isolate,expand and also calculate the frequency of neural stem cells (NSCs). Furthermore,this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied,overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay,the neural-colony forming cell assay (N-CFCA),which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential,and thus provides a method to enumerate NSC frequency. In the N-CFCA,colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC,while colonies textless 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are textless 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated. View Publication

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1),suggesting a role in neutrophil migration. However,CD177pos neutrophils exhibit no clear migratory advantage in vivo,despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system,we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils,an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly,CD177 ligation enhanced its interaction with β2 integrins,as revealed by fluorescence lifetime imaging microscopy,leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity,impaired internalization of integrin attachments,and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. View Publication

BACKGROUND CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here,we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy. METHODS Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry,also in the presence of selective inhibitors of CD73. Interferon (IFN)-$\gamma$ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control. RESULTS Isolated exosomes carried CD73 on their surface,which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-$\gamma$ release,which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment,expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders,while exosomal CD73 resulted significantly increased in non-responders. CONCLUSIONS CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment,elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy. View Publication

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including,synthetic and structural biology,cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity,thus impeding unrestricted multigene expression. We developed MultiPrime,a modular,non-cytotoxic,non-integrating,baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types,including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming,and for genome editing and engineering by CRISPR/Cas9. Moreover,we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool,to deliver multiple genes for a wide range of applications in primary and established mammalian cells. View Publication

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However,current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel,humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy,left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery,patient received multiple lines of standard of care therapy including sunitinib,axitinib,bevacizumab,everolimus and cabozantinib. While progressing on cabozantinib,he was treated with nivolumab. Seven years after initiation of nivolumab,and 4 years after stopping systemic therapy,he remains in complete remission. To generate autologous PDX model,bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant,the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore,HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells,B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood,our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting. View Publication

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level,it results in drastic chromatin changes to erase the existing somatic memory" and to establish the pluripotent state. Accordingly� View Publication

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases,and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities,the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are,thus,of interest,as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However,characterization of such MSC-like cells is currently inadequate,especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study,we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs),and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile,and they were capable of differentiation in vitro along the osteogenic,chondrogenic,and adipogenic lineages. However,compared with the parental BMSCs,iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We,therefore,conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics. View Publication

The Kit receptor functions in hematopoiesis,lymphocyte development,gastrointestinal tract motility,melanogenesis,and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo,we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice,lymphopoiesis is affected differentially. The KitY567F mutation,but not the KitY719F mutation,blocks pro T cell and pro B cell development in an age-dependent manner. Thus,the Src family kinase,but not the PI 3-kinase docking site in Kit,mediates a critical signal for lymphocyte development. In agreement with these results,treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development,similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore,Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo. View Publication

After DNA replication,sister chromatids must be untangled,or decatenated,before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation,causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint,and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain,and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance,Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro,purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus,Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors,and Metnase is a potential therapeutic target for small molecule interference. View Publication

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC,the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming,low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo,as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice,but not in high affinity-primed mice. Mechanistically,low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge,low affinity-primed CD45RB(hi) cells became CD45RB(lo),demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus,these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency,low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. View Publication

AbstractObjectiveNatural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However,clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells,resulting in the generation of supercharged NK (sNK) cells.Methods and analysisWe explored the proteomic,transcriptomic and functional characterisation of sNK cells using cell imaging,flow cytometric analysis,51-chromium release cytotoxicity assay,ELISA,ELIspot,IsoPLexis single-cell secretome analysis,proteomic analysis,RNA analysis,western blot and enzyme kinetics.ResultsWe found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1,JUN,BHLHE40,ELF1,MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation,respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.ConclusionsThese results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process. View Publication