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

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3,while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1,cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] textless 500/microL) and thrombocytopenia (THROM; platelet count textless 20,000/microL) were assessed. Synthokine significantly (P textless .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered,the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine,SC55 94,administered therapeutically post-TBI,significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host. View Publication

Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs),but activation of progenitor cells (HPCs). We identify components of the transforming growth factor beta$ (TGFbeta$) pathway as key targets of miR-143/145. Enforced expression of the TGFbeta$ adaptor protein and miR-145 target,Disabled-2 (DAB2),recapitulates the HSC defect seen in miR-143/145-/- mice. Despite reduced HSC activity,older miR-143/145-/- and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy,associated with expansion of HPC. Thus,miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFbeta$/DAB2 activation,and loss of these miRNAs creates a preleukemic state. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

Mature erythrocytes are known to lack major histocompatibility complex (MHC) proteins. However,the presence of MHC molecules on erythrocytes has been occasionally reported,though without a defined function. In this study,we designed erythrocyte conjugated solely with a fusion protein consisting of an antigenic peptide linked to MHC class I (MHC-I) protein,termed MHC-I‒Ery. The modified erythrocyte,decorated with the peptide derived from human papillomavirus (HPV) 16 oncoprotein E6/E7,effectively activated antigen-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from HPV16+ cervical cancer patients. Additionally,MHC-I‒Ery monotherapy was shown to inhibit antigen-positive tumor growth in mice. This treatment immediately activated CD8+ T cells and reduced suppressive myeloid cells in the spleen,leading to systemic anti-tumor activity. Safety and tolerability evaluations of MHC-I‒Ery in non-human primates further supported its clinical potential. Our results first demonstrated that erythrocytes equipped solely with antigen peptide‒MHC-I complexes can robustly stimulate the immune system,suggesting a novel and promising approach for advancing cancer immunotherapy. View Publication

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers,sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors,RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines,co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines,transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition,a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines,with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6,whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines,acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall,this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. View Publication

Acute and chronic solid organ failures are costly disease processes with high mortality rates. Inflammation plays a central role in both acute and chronic organ failure,including heart,lung and kidney. In this regard,new therapies for these disorders have focused on inhibiting the mediators of inflammation,including cytokines and free radicals,with little or no success in clinical studies. Recent novel treatment strategies have been directed to cell-based rather than mediator-based approaches,designed to immunomodulate the deleterious effects of inflammation on organ function. One approach,cell therapy,replaces cells that were damaged in the acute or chronic disease process with stem/progenitor technology,to rebalance excessive inflammatory states. As an example of this approach,the use of an immunomodulatory role of renal epithelial progenitor cells to treat acute renal failure (ARF) and multiorgan failure arising from acute kidney injury is reviewed. A second therapeutic pathway,cell processing,does not incorporate stem/progenitor cells in the device,but rather biomimetic materials that remove and modulate the primary cellular components,which promote the worsening organ tissue injury associated with inflammation. The use of an immunomodulating leukocyte selective cytopheretic inhibitory device is also reviewed as an example of this cell processing approach. Both of these unconventional strategies have shown early clinical efficacy in pilot clinical trials and may transform the therapeutic approach to organ failure disorders. View Publication

CRISPR-Cas9-mediated gene editing has vast applications in basic and clinical research and is a promising tool for several disorders. Our lab previously developed a non-integrating RNA virus,measles virus (MeV),as a single-cycle reprogramming vector by replacing the viral attachment protein with the reprogramming factors for induced pluripotent stem cell generation. Encouraged by the MeV reprogramming vector efficiency,in this study,we develop a single-cycle MeV vector to deliver the gRNA(s) and Cas9 nuclease to human cells for efficient gene editing. We show that the MeV vector achieved on-target gene editing of the reporter (mCherry) and endogenous genes (HBB and FANCD1) in human cells. Additionally,the MeV vector achieved precise knock-in via homology-directed repair using a single-stranded oligonucleotide donor. The MeV vector is a new and flexible platform for gene knock-out and knock-in modifications in human cells,capable of incorporating new technologies as they are developed. Graphical abstract Devaux and colleagues developed a novel single-cycle measles vector allowing gene editing of human cells. They show that Measles can express the CRISPR-Cas9 and gRNA from one genome. Finally,they demonstrate that these vectors can efficiently perform KO and knock-in in human cells without excessive off-target effects. View Publication

A network of transcription factors (TFs) determines cell identity,but identity can be altered by overexpressing a combination of TFs. However,choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of 2,000 TFs. Here,we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1,Mef2c,Esx1,Foxa1,Hnf4a,Gata2,Gata3,Myc,Elf5,Irf2,Elf1,Sfpi1,Ets1,Smad7,Nr2f1,Sox11,Dmrt1,Sox9,Foxg1,Sox2,or Ascl1-can direct efficient,specific,and rapid differentiation into myocytes,hepatocytes,blood cells,and neurons. Furthermore,transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation,and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates. View Publication

SUMMARY Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA,which may comprise cellular debris and pathogen genomes. Here we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid,energy-dependent,and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake,with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1,toll-like receptors,and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs,a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an “eat me” signal and danger-associated molecular pattern,indicating orderly pathways of recognition and disposal. eTOC Blurb: Amaya et. al. explores how cells take up extracellular circular RNAs (CircRNAs) and their impact on immune signaling. Macrophages readily internalize circRNAs,and this study identifies the specific receptors and signaling pathways governing circRNA internalization,highlighting their role as signaling molecules for immune recognition and disposal. Graphical Abstract View Publication