FACS-based Method Research Articles

CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

Deep Characterization of Human γδ T Cell Subsets Defines Lineage-Specific Traits

Abstract Human γδ T cells represent a small fraction of T cells in peripheral blood (1–10%). They constitute a unique subset of T lymphocytes that recognize non-peptide antigens through MHC-independent presentation. Major γδ T cell subsets, Vδ1 and Vδ2 possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that Vδ1 and Vδ2 T cells possess differences at the gene, phenotypic, and functional level that distinguish them as distinct lineages. Comparisons between each subset have been limited by challenges in isolating sufficient cell numbers for characterization. Through multiparametric flow cytometry we identified phenotypic signatures that defined memory and tissue homing properties specific for each subset. The immune checkpoint marker TIGIT marked a Vδ1 T cell population enriched for higher cytotoxic marker expression. Using a stringent FACS-based isolation method, we compared cytotoxicity and gene expression profiles of highly purified human Vδ1 and Vδ2 cells. Cytotoxicity assays revealed similar killing capacity of Daudi cells possibly through dissimilar mechanisms. Microarray analysis identified shared and subset-specific genetic programs including lower expression of CD3ζ in Vδ1 T cells. Analysis of transcription factors associated with subset-specific gene profiles implicated NFAT in Vδ1 and AhR in Vδ2 T cells as key regulators of ex vivo gene expression. In confirmatory assays, using TCR-dependent and independent stimuli we show higher sensitivity to calcium-mediated NFAT activity in Vδ1 T cells while Vδ2 T cells may require additional activating stimuli. These findings will facilitate further studies into the specific contributions of Vδ1 and Vδ2 T cells to the immune system. NIH NIAID R01 AI125097

Deep characterization of human γδ T cell subsets defines shared and lineage-specific traits.

Under non-pathological conditions, human γδ T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses.

Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method.

Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of complex oligosaccharides has been of great scientific and industrial interest. Here we describe an ultra-high-throughput fluorescence activated cell sorting (FACS) method for the directed evolution of GTs, at the single cell level. This assay relies on the exquisite substrate specificity of lactose permeases (LacY) that are located in the cell membrane, which distinguish selectively the fluorescent glycosylated products from the unreacted substrates. The method described here allows facile screening 106-107 mutants per hour. We demonstrate the application of this technique in the screening of libraries of α1,3-fucosyltransferase.

A MULTIMODAL APPROACH FOR AUTOMATIC DETECTION OF INFANT PAIN USING FACIAL EXPRESSION AND CRYING

research on automatic detection of facial expressions has been making great strides over the past decade. One challenge in this area has been in not just recognizing the regular facial expressions but in paying attention to the micro expressions or compound expressions i.e. combining multiple expressions which may be critical to the success of an automated system. For instance, detection of pain/no-pain would be of great benefit especially in case of infant pain recognition. This is because the current standard for assessing neonatal pain is discontinuous and inconsistent as it depends highly on the observer’s bias. And sometimes, the drawbacks of manual pain assessment can result in delayed intervention and inconsistent treatment of pain. Hence, there is a need for machine-based techniques to provide consistent and continuous automatic assessment. Preprocessing, pain analysis or feature extraction, and pain recognition are the three main phases in automatic pain detection. Pain recognition from facial gestures is a specific task within the broader task of facial expression recognition i.e. Pain is a compound expression derived from many facial gestures. Hence, pain detection is a very important task from the point of view of computer vision, since it is a clear step towards automatic detection of spontaneous face expressions. The vast majority of the existing methods in the field of automatic facial expressions recognition use Facial Action Coding System (FACS) to detect facial expressions of only adults. However, literature shows that there is ample scope for research in FACS-based method that is designed specifically to detect infants’ facial expression of pain. Furthermore, the challenge in Facial Expression Recognition of Infant pain is because of the fact that the infants’ facial expressions include additional movements and units that are readily not available in the FACS. The purpose of this paper is to conduct a literature survey on the existing research in the area of automated pain analysis and recognition and facilitate in the development of a multi modal pain assessment system for children and infants. Further, this paper discusses how machine learning methods have been used in the Automatic detection of Pain for improving the overall performance. Techniques like Convolutional neural networks (CNNs) and Long-Short Term Memory (LSTM) networks have gained much popularity in the last decades due to the wide range of their applications in medical image analysis and emotion recognition.

FACS isolation of low percentage human antigen-specific CD8+ T cells based on activation-induced CD3 and CD8 downregulation

As T cell activation leads to downregulation of T cell receptor (TCR) and coreceptor CD8, we developed a novel FACS-based sorting method to enrich activated antigen-specific CD8+ T cells. Using multiple established or low percentage T cell cultures, with either single antigen specificity or multiple influenza A virus antigen specificities, we have optimized the sorting method for T cell activation time and stimulating antigen dose. We have also sorted various numbers of antigen-specific CD8+ T cells into 96-well plates to demonstrate these T cells are capable of expanding into nearly pure CD8+ T cell lines. Our approach has the advantage of sorting antigen-specific T cells without knowing their specific antigenic epitopes or restricting HLA. We believe this method can be very helpful for successfully establishing CD8+ T cell lines for various purpose, including immunotherapy.

Fluorescence Activated Cell Sorting (FACS) in Genome-Wide Genetic Screening of Membrane Trafficking.

About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.

Mroh1, a lysosomal regulator localized by WASH-generated actin.

ABSTRACTThe steps leading to constitutive exocytosis are poorly understood. In Dictyostelium WASH complex mutants, exocytosis is blocked, so cells that take up fluorescent dextran from the medium retain it and remain fluorescent. Here, we establish a FACS-based method to select cells that retain fluorescent dextran, allowing identification of mutants with disrupted exocytosis. Screening a pool of random mutants identified members of the WASH complex, as expected, and multiple mutants in the conserved HEAT-repeat-containing protein Mroh1. In mroh1 mutants, endosomes develop normally until the stage where lysosomes neutralize to postlysosomes, but thereafter the WASH complex is recycled inefficiently, and subsequent exocytosis is substantially delayed. Mroh1 protein localizes to lysosomes in mammalian and Dictyostelium cells. In Dictyostelium, it accumulates on lysosomes as they mature and is removed, together with the WASH complex, shortly before the postlysosomes are exocytosed. WASH-generated F-actin is required for correct subcellular localization; in WASH complex mutants, and immediately after latrunculin treatment, Mroh1 relocalizes from the cytoplasm to small vesicles. Thus, Mroh1 is involved in a late and hitherto undefined actin-dependent step in exocytosis.

Selection of High-Producing Clones Using FACS for CHO Cell Line Development.

Cell line development aims to generate and select clones with desirable characteristics. One of the most important parameters for biopharmaceutical cell selection is cell-specific productivity (Qp) or the quantity of product produced per cell per day. Fluorescence-activated cell sorting (FACS) is a powerful, high-throughput technique that facilitates multiparametric characterization and isolation of individual cell clones from heterogeneous populations. Here, we describe a FACS-based method for section of high-producing CHO cell clones.

High TCR Vbeta Skewing in CD4+ and Low in CD8+ Is Associated with Development of Chronic Graft-Versus-Host Disease

IntroductionChronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Perturbations in immune reconstitution following allo-HSCT are believed to be integral to the pathophysiology of cGVHD. We hypothesized that skewing in T cell repertoire around day 80 would be predictive of cGVHD, while maintenance of a broad repertoire would be protective. One method used to assess skewing is the Gini Index, a conventional measure of income inequality. In this prospective study, we utilized a rapid FACS based method of TCR Vβ repertoire analysis within CD4+ and CD8+ in patients at day 80-110 post allo-HSCT to determine whether preferential TCR skewing predicts the development of cGVHD.MethodsPeripheral blood samples were obtained from 79 consented patients at day 80-110 following allo-HSCT at The University of Texas MD Anderson Cancer Center from October 2012 to November 2015. All patients were in remission of their disease at the time of blood sampling. Fresh PBMC were analyzed by FACS after staining for CD3, CD4, CD8 and IOTest Beta Mark 24 TCR Vβ kit. The TCR Gini Index scores (GS) were calculated for the T cell subsets, and ranged from 0 to 100. Low GS indicate more equal distribution of TCR-Vβ families (i.e. broad repertoire), whereas high GS reflect unequal distribution of TCR-Vβ families (i.e. repertoire skewing). Predictive thresholds of GS were identified in quartiles analysis and confirmed by Receiver Operating Characteristic (ROC) curve.The association between GS and the rate of cGVHD since the date of blood sampling was evaluated using competing-risks regression analysis. The diagnosis of cGVHD was based on the 2014 National Institutes of Health guidelines.ResultsThe median age at transplant was 53 years (range 21-75) and 60% of patients were male. The most common transplant indication was AML/MDS (52%) followed by lymphoma (16%), ALL (14%), and other (18%). Graft source was peripheral blood (54%), bone marrow (43%) and umbilical cord blood (UCB) (3%) with donors being matched unrelated (65%), matched-related (24%), mismatch related (9%) and UCB (3%). Forty three (54%) patients were not in remission at the time of transplant. At the time of enrollment, 47% had history of grade II-IV acute GVHD and 44% were on systemic steroids. At a median follow up of 2 years since blood sampling, 16 patients developed cGVHD (cumulative incidence (CI) 23%; 95% Confidence Interval: 15-35). The median time from HSCT to cGVHD was 174 days (range 111-551 days). None of the clinical or demographic characteristics listed above, except history of acute GVHD, was significantly associated with the incidence of cGVHD. GS were significantly predictive of the incidence of cGVHD. The 2 year %CI of cGVHD was 47% in patients who had CD4+ TCR GS >55% (n=19) compared with 16% in those with CD4+ GS ≤55% (n=60) (hazard ratio (HR) =3.4, P=0.01). In contrast to CD4+, lower CD8+ GS predicted for higher incidence of cGVHD. The 2 year %CI of cGVHD was 39% in patients (n=34) with CD8+ GS ≤60% compared with 10% in patients (n=45) with CD8+ GS >60% (HR=4.5, P=0.009). When both CD4+ and CD8+ GS were considered in combination, the 2 year incidence of cGVHD was highest in patients (n=6) with both low CD8+ (≤60%) and high CD4+ (>55%) GS, and lowest in patients (n=32) with high CD8+ (>60%) and low CD4+ (≤55%) GS (%CI: 83% vs 4%, P=0.001). The combined effect of high CD4+ and low CD8+ TCR skewing was more pronounced in patients with a history of grade II-IV acute GVHD. Low CD8+ (≤60%, n=28) or high CD4+ (>55%, n=13) GS occurring separately were associated with comparable rates of cGVHD (%CI: 31% and 27%, p=0.8), irrespective of history of grade II-IV acute GVHD (Figure).ConclusionsHigh CD4+ and low CD8+ TCR Gini indices were independent predictors of cGVHD. Our results indicated that the higher skewing of CD8+ TCR appeared to be protective of cGVHD and the opposite for CD4+. Our study is the first to test the usefulness of FACS-based TCR measurement and Gini Index score in a clinical setting to help predict the development of cGVHD and provide greater insights into the pathogenesis of cGVHD. Using this convenient FACS-based method to identify patients who are at risk of developing cGVHD could be beneficial from development of effective preventive or therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

Detection of molecular alterations in methamphetamine‐activated Fos‐expressing neurons from a single rat dorsal striatum using fluorescence‐activated cell sorting (FACS)

Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence-activated cell sorting (FACS)-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5-6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as five Fos-positive neurons. Methamphetamine induced 3-20-fold increases of immediate early genes arc, homer-2, c-fos, fosB, and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not Fos-negative neurons. Immediate early gene mRNA induction was 10-fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models. Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre-amplification to assess acute methamphetamine-induced gene expression from as few as 5 Fos-expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3-20-fold increases of immediate early genes (IEGs) in Fos-positive but not Fos-negative neurons. Targeted PCR pre-amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.

Pancreatic cancer circulating tumour cells express a cell motility gene signature that predicts survival after surgery.

BackgroundMost cancer deaths are caused by metastases, resulting from circulating tumor cells (CTC) that detach from the primary cancer and survive in distant organs. The aim of the present study was to develop a CTC gene signature and to assess its prognostic relevance after surgery for pancreatic ductal adenocarcinoma (PDAC).MethodsNegative depletion fluorescence activated cell sorting (FACS) was developed and validated with spiking experiments using cancer cell lines in whole human blood samples. This FACS-based method was used to enrich for CTC from the blood of 10 patients who underwent surgery for PDAC. Total RNA was isolated from 4 subgroup samples, i.e. CTC, haematological cells (G), original tumour (T), and non-tumoural pancreatic control tissue (P). After RNA quality control, samples of 6 patients were eligible for further analysis. Whole genome microarray analysis was performed after double linear amplification of RNA. ‘Ingenuity Pathway Analysis’ software and AmiGO were used for functional data analyses. A CTC gene signature was developed and validated with the nCounter system on expression data of 78 primary PDAC using Cox regression analysis for disease-free (DFS) and overall survival (OS).ResultsUsing stringent statistical analysis, we retained 8,152 genes to compare expression profiles of CTC vs. other subgroups, and found 1,059 genes to be differentially expressed. The pathway with the highest expression ratio in CTC was p38 mitogen-activated protein kinase (p38 MAPK) signaling, known to be involved in cancer cell migration. In the p38 MAPK pathway, TGF-β1, cPLA2, and MAX were significantly upregulated. In addition, 9 other genes associated with both p38 MAPK signaling and cell motility were overexpressed in CTC. High co-expression of TGF-β1 and our cell motility panel (≥ 4 out of 9 genes for DFS and ≥ 6 out of 9 genes for OS) in primary PDAC was identified as an independent predictor of DFS (p=0.041, HR (95% CI) = 1.885 (1.025 – 3.559)) and OS (p=0.047, HR (95% CI) = 1.366 (1.004 – 1.861)).ConclusionsPancreatic CTC isolated from blood samples using FACS-based negative depletion, express a cell motility gene signature. Expression of this newly defined cell motility gene signature in the primary tumour can predict survival of patients undergoing surgical resection for pancreatic cancer.Trial RegistrationClinical trials.gov NCT00495924

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

BackgroundThe establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production.ResultsAn internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb) than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones.ConclusionsThis study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

A Novel Method for Isolating Individual Cellular Components from the Adult Human Distal Lung

A variety of lung diseases, such as pulmonary emphysema and idiopathic pulmonary fibrosis, develop in the lung alveoli. Multiple cell types are localized in the alveoli, including epithelial, mesenchymal, and endothelial cells. These resident cells participate in the pathogenesis of lung disease in various ways. To elaborate clearly on the mechanisms of these pathologic processes, cell type-specific analyses of lung disease are required. However, no method exists for individually isolating the different types of cells found in the alveoli. We report on the development of a FACS-based method for the direct isolation of individual cell types from the adult human distal lung. We obtained human lung tissue from lung resections, and prepared single-cell suspension. After depleting CD45-positive cells, a combination of antibodies against epithelial cell adhesion molecule (EpCAM), T1α, and vascular endothelial (VE)-cadherin as used to delineate alveolar cell types. Alveolar Type II cells were highly purified in the EpCAM(hi)/T1α(-) subset, whereas the EpCAM(+)/T1α(-/low) subset contained a mixed epithelial population consisting of alveolar Type I and bronchiolar epithelial cells. The EpCAM(-)/T1α(-) subset included both microvascular endothelial and mesenchymal cells, and these were separated by immunoreactivity to VE-cadherin. Lymphatic endothelial cells existed in the EpCAM(-)/T1α(hi) subset. Isolated cells were viable, and further cell culture studies could be performed. These results suggest that this novel method enables the isolation of different cellular components from normal and diseased lungs, and is capable of elucidating phenotypes specific to certain alveolar cell types indicative of lung disease.

Stress Erythropoiesis: HIF-1 Alpha Synergizes with Glucocorticoids to Induce BFU-E Progenitor Self-Renewal

AbstractAbstract 814The goal of this study was to find small molecules that stimulate erythropoiesis at an earlier stage than erythropoietin (Epo). We therefore studied the mechanisms by which glucocorticoids (GCs) promote erythroblast formation during stress erythropoiesis (SE). Since the target cell of GCs in SE was not known we first established a FACS-based method to purify burst-forming unit erythroid (BFU-E) and colony-forming unit of erythroid lineage (CFU-E) cells from mouse fetal liver. BFU-E and CFU-E cells were resolved by differences in CD71 or CD24a expression (BFU-E = CD71/CD24a10% low; CFU-E = CD71/CD24a20% high) from a population of cells positive for Kit, and negative for the cell surface markers Ter119, B220, Mac-1, CD3, Gr-1, Sca-1, FcGR, CD41 and CD34. Purified “BFU-E” cells formed 94% BFU-E colonies and “CFU-E” cells formed 95% CFU-E colonies at cloning efficiencies of 55–70%. Using pure CFU-E and BFU-E progenitors we demonstrated that while 100nM Dexamethasone (Dex) does not increase proliferation or self-renewal of CFU-E cells, Dex stimulates the earlier BFU-E progenitors to undergo limited self-renewal, which increases formation of CFU-E cells and erythroblasts >20-fold. After identifying BFU-E progenitors as the target cells of GCs during SE, we next determined how DEX changes miRNA and mRNA expression in BFU-Es by next generation sequencing. No miRNAs changed the level of expression more than 50%. Of the 10,000 most expressed mRNAs 83 were up-regulated and 112 mRNAs down-regulated more than 2-fold in response to Dex. Since previous studies show that dimerization (trans activation) of the GC receptor is necessary for GCs to induce erythroblast production, the most important GC target genes are likely up- rather than down-regulated. We hypothesized that other molecules that are able to enhance the expression of the 83 up-regulated genes could enhance or replace the stimulatory effect of Dex. We therefore computationally analyzed the promoter regions of the up-regulated genes using motif enrichment analysis. We found that Dex induces expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1 alpha (HIF1a) binding sites (p-value = 1e–27). Analysis of six published gene expression datasets, where the effect of GCs on gene expression had been determined in cell types other than BFU-E cells, showed very low or no enrichment of HIF1a targets among genes up-regulated by Dex. We confirmed the suggested overlapping effects of HIF1a and GCs on gene expression in BFU-E cells by comparing the effect of Dex with that of pharmacological HIF1a activation by the prolyl hydroxylase inhibitor (PHI) Dimethyloxalylglycine (DMOG). As determined by next generation mRNA sequencing, DMOG increased expression of 98 genes more than 50%, of which 28 were among 109 genes up-regulated >50% by Dex (p-value for overlap = 1e–26). Importantly the overlap in effects on gene expression also resulted in overlapping biological effects. Similar to Dex, DMOG had little effect on CFU-E cells (Figure A). In contrast DMOG synergized with Dexamethasone to promote BFU-E self-renewal and prevent BFU-E cell exhaustion, enhancing the production of CFU-E cells 170-fold and maximum erythroblast production 300-fold (Figure B). DMOG thus enhanced the stimulatory effect of 100nM Dex on erythroblast production 7-fold. In similar experiments where BFU-E cells were cultured in GCs levels corresponding to physiological cortisol levels (1 nM Dex), DMOG enhanced erythroblast production 10-fold. We further showed that DMOG and Dex also synergistically enhance proliferation of adult mouse and human erythroid progenitors. Our results support a physiological model of SE where increased systemic levels of free cortisol, in combination with local anoxia, induce BFU-E self-renewal and increase erythroblast production. This suggests that the therapeutic potential for PHIs in treatment for anemia goes beyond replacing recombinant Epo. Here we show that in addition to enhancing Epo production from the kidneys, PHIs have an intrinsic stimulatory effect on BFU-E progenitors, leading to increased production of Epo-responsive CFU-E cells. Thus PHIs provide a new window for treating Epo-resistant anemia. The synergistic effect of GCs and PHIs further suggests that PHIs may enhance or replace the effect of Prednisone in the treatment of Diamond-Blackfan Anemia and other bone marrow failure syndromes. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

10-color FACS-based method of chimerism analysis after multi-cord blood transplantation

10-color FACS-based method of chimerism analysis after multi-cord blood transplantation

Apoptotic effects of Human Herpesvirus-6A on glia and neurons as potential triggers for central nervous system autoimmunity

Background Human Herpesvirus type 6 (HHV-6A and/or HHV-6B) has been tentatively associated with multiple sclerosis (MS). However, there is currently no direct proof of pathogenicity. Objectives To determine whether exposure to HHV-6 variants is capable of inducing programmed cell death (apoptosis) in representative cell types of the central nervous system (CNS). Study design HHV-6A and HHV-6B variants were grown on human T cell lines HSB2 and MOLT-3, respectively. Human neuronal (SK-N-SH), astrocytes (CRT), and oligodendrocytes (TC620) cell lines were exposed in vitro to infected T cells in a trans-well system for up to 4 days (5×10 4 cells target cells and 2×10 6 T cells). Apoptosis was measured by a FACS-based method. Results Exposure to HHV-6A induced apoptosis in a time-dependent manner, while exposure to HHV-6B did not. Three days after exposure, apoptosis was increased compared to normalized controls, by 239% in neurons, 321% in astrocytes, and 326% in oligodendrocytes, respectively. Conclusions This study provides the demonstration that exposure to immune cells carrying replicating HHV-6A may injure glial cells and neurons by inducing apoptosis, and direct evidence for a causal association between HHV-6A with MS and related disorders.

A rapid method for the quantification of mouse hippocampal neurogenesis in vivo by flow cytometry : Validation with conventional and enhanced immunohistochemical methods

Neural stem cells reside in the subventricular zone and the dentate gyrus of the hippocampus in adult mammalian brain. In the hippocampus, a number of factors are reported to modulate the rate of neural progenitor proliferation in the hippocampus, such as exercise, corticosteroids, and many pharmacological agents including several classes of antidepressants. It is currently unclear whether this increased proliferation is physiologically relevant, but it provides a potentially useful biomarker to assess novel antidepressant compounds. Changes in neurogenesis are typically quantified by administration of bromodeoxyuridine (BrdU) in vivo, and subsequent quantification of labelled nuclei. A robust and rapid means of quantifying BrdU labelling in adult hippocampus in vivo would allow higher throughput screening of potential antidepressant compounds. In this study we describe a FACS-based method for quantification of BrdU labelled cells in fixed cell suspensions from BrdU-treated adult mouse hippocampus. A variety of experimental conditions known to modulate proliferation were tested, including administration of corticosterone and the antidepressants imipramine and fluoxetine. The robust changes compared to control groups observed in these models were similar to previously reported studies, thus offering a more rapid and streamlined means to quantify effects of compounds on hippocampal proliferation.

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

BackgroundPoly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions.ResultsThis paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay.ConclusionThe methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type.