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

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche,but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor,RET,at the cell surface of HSCs,in mediating sustained cellular growth,resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex,GDNF/GFRa1,show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally,we demonstrate that RET drives a multi-faceted intracellular signalling pathway,including key signalling intermediates AKT,ERK1/2,NFkB and p53,responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions. 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

Realizing the potential that human embryonic stem cells (hESCs) hold,both for the advancement of biomedical science and the development of new treatments for many human disorders,will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard,it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay,thus,provides a valuable tool for monitoring the quality of hESC cultures,and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies),or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm,mesoderm,and ectoderm lineages. 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

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

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

In human cytomegalovirus (HCMV)-seropositive patients,CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However,analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors,availability of cells,and low frequency of reactivation. In addition,multiple progenitor cell types express surface CD34,and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study,we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines,WA01 and WA09,to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however,these cells are a heterogeneous pool with donor-to-donor variation in functional,genetic,and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states,which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation. View Publication

SummaryToxic signaling by extrasynaptic NMDA receptors (eNMDARs) is considered an important promoter of amyotrophic lateral sclerosis (ALS) disease progression. To exploit this therapeutically,we take advantage of TwinF interface (TI) inhibition,a pharmacological principle that,contrary to classical NMDAR pharmacology,allows selective elimination of eNMDAR-mediated toxicity via disruption of the NMDAR/TRPM4 death signaling complex while sparing the vital physiological functions of synaptic NMDARs. Post-disease onset treatment of the SOD1G93A ALS mouse model with FP802,a modified TI inhibitor with a safe pharmacology profile,stops the progressive loss of motor neurons in the spinal cord,resulting in a reduction in the serum biomarker neurofilament light chain,improved motor performance,and an extension of life expectancy. FP802 also effectively blocks NMDA-induced death of neurons in ALS patient-derived forebrain organoids. These results establish eNMDAR toxicity as a key player in ALS pathogenesis. TI inhibitors may provide an effective treatment option for ALS patients. Graphical abstract Highlights•eNMDARs promote ALS disease progression via the NMDAR/TRPM4 death signaling complex•TwinF interface inhibitor FP802 disrupts the NMDAR/TRPM4 death signaling complex•FP802 is therapeutically effective in an ALS mouse model•FP802 protects against NMDA-induced death in brain organoids from ALS patient iPSCs Yan et al. find that FP802,which provides neuroprotection by detoxifying eNMDARs through disruption of the NMDAR/TRPM4 complex,halts motor neuron loss in an ALS mouse model,reduces serum NfL levels,improves motor performance,and extends life expectancy. FP802 is also neuroprotective in brain organoids derived from ALS patients. View Publication

ABSTRACTDuchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration,regeneration,and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches,including exon skipping and gene replacement therapy,are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo,HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However,HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT,demonstrating a strain-specific difference in DMD-related immunopathology. Summary: Duchenne muscular dystrophy is a devastating that currently has limited treatment options. RNA sequencing and downstream analysis in iSkM and mdx samples revealed HMGB1 may be a relevant treatment biomarker. View Publication