Differences In Surface Marker Expression Research Articles

Neutrophil Inhibitory Receptors Expression Is Distinct During Gram-Positive and -Negative Pneumonia and Affects Neutrophil Function

Abstract Rational: Despite effective antimicrobial therapy, morbidity and mortality from pneumonia remains high. Honing our understanding of immune dysfunction in pneumonia is now crucial for developing targeted therapeutics. Our objective is to understand how different lethal bacterial pneumonias affect neutrophil phenotype and function over time and identify regulators of neutrophil function. Methods: Mice were intratracheally instilled with E. colior S. pneumoniae(SP3) for 6, 24 or 48h and neutrophils were isolated from either bronchioloalveolar lavage fluid (BALF) or peripheral blood. Neutrophil surface marker expression (SME) was analyzed via 25-color panel run on a Cytek Aurora spectral flow cytometer (SFC). Data was analyzed in FlowJo and gated for live, single cell, CD45+Ly6G+ neutrophils, opt-SNE was run for data visualization, and Phenograph was run for unbiased clustering. For bacterial clearance assays, bone marrow neutrophils were isolated via Percoll, then incubated with blocking antibodies against inhibitor receptors (IRs) (PD-L1, SIRPα, VISTA, or CD200R), soluble ligands, or media only. Neutrophils were then cocultured with either SP3 or E. coli. Media from bacteria-neutrophil cultures was then plated on agar and the developing colonies were counted. Results and Conclusions: Using SFC, we identified pathogen- and timepoint-specific differences in SME of the IRs PD-L1, SIRPα, VISTA and CD200R. Modulating IR signaling by blocking or stimulating them impacts neutrophils’ ability to clear bacteria. In sum, our data suggests that neutrophils modulates their response to adapt to specific pathogens. IRs may play a role in regulating these dynamic neutrophil responses, making them a potential therapeutic target. NIH grants K08 HL130582 (KET) and R01 HL158732 (KET).

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags

BackgroundA major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags.MethodsMo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized.ResultsMonocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels.ConclusionsOur findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Transplantation of stem cells from umbilical cord blood as therapy for type I diabetes.

In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish β-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.

Progenitor cells from atria, ventricle and peripheral blood of the same patients exhibit functional differences associated with cardiac repair

AimDeciding the best cell type for cardiac regeneration remains a big challenge. No studies have directly compared the functional efficacy of cardiac progenitor cells (CPCs) with extra-cardiac stem cells isolated from the same patient. Methods and resultsWe compared the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CPCs and left ventricular (LV) CPCs isolated from the same patients (n=14). Within the same heart, RAA and LV CPCs exhibited marked differences in surface marker expression, with RAA CPCs exhibiting better expansion potential and migration properties. When subjected to hypoxia and serum starvation to simulate in vivo ischemic environment, RAA and LV CPCs exhibited similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under hypoxia. RT-profiler array showed comparable gene expression pattern in RAA and LV CPCs, while they were differentially expressed in EPCs. Further, treating human umbilical vein endothelial cells with conditioned medium (CM) from LV showed maximum angiogenic potential, while cardiomyocytes treated with CM from RAA showed greatest survival under hypoxic conditions. ConclusionsResults from this study provide the first evidence that progenitor cells from different regions exhibit functional differences within the same patient.

Chronic High-Fat Feeding Affects the Mesenchymal Cell Population Expanded From Adipose Tissue but Not Cardiac Atria.

Mesenchymal stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. However, in the clinic, cells will be isolated from patients who may be suffering from comorbidities such as obesity and diabetes, which are known to adversely affect progenitor cells. Here we determined the effect of a high-fat diet (HFD) on mesenchymal stem cells from cardiac and adipose tissues. Mice were fed a HFD for 4 months, after which cardiosphere-derived cells (CDCs) were cultured from atrial tissue and adipose-derived mesenchymal cells (ADMSCs) were isolated from epididymal fat depots. HFD raised body weight, fasted plasma glucose, lactate, and insulin. Ventricle and liver tissue of HFD-fed mice showed protein changes associated with an early type 2 diabetic phenotype. At early passages, more ADMSCs were obtained from HFD-fed mice than from chow-fed mice, whereas CDC number was not affected by HFD. Migratory and clonogenic capacity and release of vascular endothelial growth factor did not differ between cells from HFD- and chow-fed animals. CDCs from chow-fed and HFD-fed mice showed no differences in surface marker expression, whereas ADMSCs from HFD-fed mice contained more cells positive for CD105, DDR2, and CD45, suggesting a high component of endothelial, fibroblast, and hematopoietic cells. Both Noggin and transforming growth factor β-supplemented medium induced an early stage of differentiation in CDCs toward the cardiomyocyte phenotype. Thus, although chronic high-fat feeding increased the number of fibroblasts and hematopoietic cells within the ADMSC population, it left cardiac progenitor cells largely unaffected. Mesenchymal cells are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. In the clinic, cells are isolated from patients who may be suffering from comorbidities such as obesity and diabetes. This study examined the effect of a high-fat diet on mesenchymal cells from cardiac and adipose tissues. It was demonstrated that a high-fat diet did not affect cardiac progenitor cells but increased the number of fibroblasts and hematopoietic cells within the adipose-derived mesenchymal cell population.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.

Arthritic periosteal tissue from joint replacement surgery: a novel, autologous source of stem cells.

The overarching aim of this study is to assess the feasibility of using periosteal tissue from the femoral neck of arthritic hip joints, usually discarded in the normal course of hip replacement surgery, as an autologous source of stem cells. In addition, the study aims to characterize intrinsic differences between periosteum-derived cell (PDC) populations, isolated via either enzymatic digestion or a migration assay, including their proliferative capacity, surface marker expression, and multipotency, relative to commercially available human bone marrow-derived stromal cells (BMSCs) cultured under identical conditions. Commercial BMSCs and PDCs were characterized in vitro, using a growth assay, flow cytometry, as well as assay of Oil Red O, alizarin red, and Safranin O/Fast Green staining after respective culture in adipo-, osteo-, and chondrogenic media. Based on these outcome measures, PDCs exhibited proliferation rate, morphology, surface receptor expression, and multipotency similar to those of BMSCs. No significant correlation was observed between outcome measures and donor age or diagnosis (osteoarthritis [OA] and rheumatoid arthritis [RA], respectively), a profound finding given recent rheumatological studies indicating that OA and RA share not only common biomarkers and molecular mechanisms but also common pathophysiology, ultimately resulting in the need for joint replacement. Furthermore, PDCs isolated via enzymatic digestion and migration assay showed subtle differences in surface marker expression but otherwise no significant differences in proliferation or multipotency; the observed differences in surface marker expression may indicate potential effects of isolation method on the population of cells isolated and/or the behavior of the respective isolated cell populations. This study demonstrates, for the first time to our knowledge, the feasibility of using arthritic tissue resected during hip replacement as a source of autologous stem cells. In sum, periosteum tissue that is resected with the femoral neck in replacing the hip represents an unprecedented and, to date, unstudied source of stem cells from OA and RA patients. Follow-up studies will determine the degree to which this new, autologous source of stem cells can be banked for future use.

Eliminating the need of serum testing using low serum culture conditions for human bone marrow-derived mesenchymal stromal cell expansion

BackgroundThe conventional expansion of human mesenchymal stromal cells (hMSC) for tissue engineering or (pre-) clinical investigation includes the use of 10% fetal bovine serum (FBS). However, there exists immense lot-to-lot variability in FBS samples and time consuming as well as cost intensive lot pre-testing is essential to guarantee optimal hMSC proliferation and stem cells characteristics maintenance. Furthermore, lot-to-lot variability impedes the long-term consistency of research and comparability between research groups. Therefore, we investigated the use of defined, invariable, non-synthetic FBS in low serum culture conditions for isolation and expansion of hMSC.MethodshMSC were isolated from bone marrow in Panserin 401 supplemented with growth factors and 2% MSC-tested or non-tested, defined, invariable, non-synthetic FBS and further cultivated in vitro. The surface marker expression, differentiation capacity as well as cell proliferation and cytotoxicity was analyzed and compared between serum samples.ResultsCells isolated and cultivated with low concentrations of MSC-tested or non-tested FBS demonstrated no differences in surface marker expression or differentiation capacity. Proliferation of hMSC was equal in medium supplemented with either serum with no indication of cell death.ConclusionsThe low serum concentration in Panserin 401 supplemented with growth factors enables the use of defined, invariable, non-synthetic FBS for the isolation and expansion of hMSC. The required hMSC characteristics like surface marker expression and differentiation capacity are maintained. Importantly, no differences in the cell proliferation could be detected. Therefore, using these low-serum culture conditions, the need for lot-to-lot pre-testing of FBS usually needed for optimal hMSC expansion is abolished leading to long-term consistency and comparability of results.

Differences in Surface Marker Expression and Chondrogenic Potential among Various Tissue-Derived Mesenchymal Cells from Elderly Patients with Osteoarthritis

Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells that could potentially be used to repair injured cartilage in diseases such as osteoarthritis (OA). In this study we used bone marrow, adipose tissue from articular and subcutaneous locations, and synovial fluid samples from 18 patients with knee OA to find a suitable alternative source for the isolation of MSCs with high chondrogenic potential. MSCs from all tissues analysed had a fibroblastic morphology, but their rates of proliferation varied. Subcutaneous fat-derived MSCs proliferated faster than bone marrow- and Hoffa’s fat pad-derived MSCs, while synovial fluid-derived MSCs grew more slowly. CD36 and CD54 expression was similar across all groups of MSCs with several minor differences. High expression of these surface markers in subcutaneous fat-derived MSCs was correlated with poor differentiation into hyaline cartilage. Synovial fluid-derived MSCs presented a relatively small chondrogenic differentiation capacity while Hoffa’s fat pad-derived MSCs had strong chondrogenic potential. In conclusion, MSCs from elderly patients with OA may still display significant chondrogenic potential, depending on their origin.

Genome-Wide DNA Methylation Profiling of Hematopoietic Stem and Progenitor Cells Reveals Over-Representation of ETS Transcrition Factor Binding Sites

Abstract 211The hematopoietic system is ideal for the study of epigenetic changes in primary cells because hematopoietic cells representing distinct stages of hematopoiesis can be enriched and isolated by differences in surface marker expression. DNA methylation is an essential epigenetic mark that is required for normal development. Conditional knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment have revealed that methylation is critical for long-term renewal and lineage differentiation of hematopoietic stem cells (Broske et al 2009, Trowbridge el al 2009). To better understand the role of DNA methylation in self-renewal and differentiation of hematopoietic cells, we characterized genome-wide DNA methylation in primary cells representing three distinct stages of hematopoiesis. We isolated mouse hematopoietic stem cells (HSC; Lin- Sca-1+ c-kit+), common myeloid progenitor cells (CMP; Lin- Sca-1- c-kit+), and erythroblasts (ERY; CD71+ Ter119+). Methyl Binding Domain Protein 2 (MBD2) is an endogenous reader of DNA methylation that recognizes DNA with a high concentration of methylated CpG residues. Recombinant MBD2 enrichment of DNA followed by massively-parallel sequencing was used to map and compare genome-wide DNA methylation patterns in HSC, CMP and ERY. Two biological replicates were sequenced for each cell type with total read counts ranging from 32,309,435–46,763,977. Model-based analysis of ChIP Seq (MACS) with a significance cutoff of p<10−5 was used to determine statistically significant peaks of methylation in each replicate. Globally, the number of methylation peaks was highest in HSC (85,797peaks), lower in CMP (50,638 peaks), and lowest in ERY (27,839 peaks). Comparison of the peaks in HSC, CMP and ERY revealed that only 2% of the peaks in CMP or ERY are absent in HSC indicating that the vast majority of methylation in HSC is lost during differentiation. Comparison of methylation with genomic features revealed that CpG islands associated with promoters are hypomethylated, while many non-promoter CpG islands are methylated. Furthermore, methylation of non-promoter associated CpG islands occurs infrequently in cell-type specific peaks but is more abundant in common methylation peaks. When the DNA methylation patterns were compared to mRNA expression, we found that as expected, proximal promoter sequences of expressed genes were hypomethylated in all three cell types, while methylation in the gene body positively correlated with gene expression in HSC and CMP. Utilizing de novo motif discovery we found a subset of transcription factor consensus binding motifs that were overrepresented in methylated sequences. Motifs for several ETS transcription factors, including GABPalpha and ELF1 were found to be overrepresented in cell-type specific as well as common methylated regions. Other transcription factor consensus sites, such as the NFAT factors involved in T-cell activation, were specifically overrepresented in the methylated promoter regions of CMP and ERY. Comparison of our methylation data with the occupancy of hematopoietic transcription factors in the HPC7 cell line, which is similar to CMP (Wilson et al 2010), revealed a significant anti-correlation between DNA methylation and the binding of Fli1, Lmo2, Lyl1, Runx1, and Scl.Our genome-wide survey provides new insights into the role of DNA methylation in hematopoiesis. Firstly, the methylation of CpG islands is associated with the most primitive hematopoietic cells and is unlikely to drive hematopoietic differentiation. We feel that the elevated genome-wide DNA methylation in HSC compared to CMP and ERY, combined with the positive association between gene body methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSC. Finally, the finding that transcription factor binding sites are over represented in the methylated sequences of the genome leads us to conclude that DNA methylation modulates key hematopoietic transcription factor programs that regulate hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Defective Macrophage Function in Crohn's Disease: Role of Alternatively Activated Macrophages in Inflammation

Introduction The aetiology of Crohn9s disease (CD) involves a genetically determined dysregulated immune response to commensal intestinal microflora. In CD, viable E coli are found within lamina propria macrophages (MΦs) and E coli intracellular survival is prolonged in CD-derived MΦs in vitro. Different MΦ subpopulations exist, M1 cells are inflammatory cells, M2a cells are involved in tissue repair and M2c are regulatory cells that limit inflammation. The role these different MΦs play in this abnormal handling of bacteria in CD is unclear. Methods The aim of this study was to examine in vitro M1 and M2 MΦ maturation in CD patients and healthy individuals and how these cells respond to challenge with CD-derived E coli. To do this we monitored MΦ morphology, surface markers and cytokine production and intracellular bacterial survival. Peripheral blood monocytes were isolated from CD patients and healthy controls and treated with cytokines to generate distinct MΦ subpopulations: IFNγ for M1, IL4/IL13 for M2a and IL10 for M2c. MΦ morphology was assessed by H&E staining and surface marker expression of CD14, CD16 and CD33, chemokine receptor CCR2, scavenger receptor CD163, co stimulatory molecule CD40 and mannose receptor CD206 using flow cytometry. IL10 and TNFα production were measured by ELISA and intracellular E coli survival was measured using the gentamicin protection assay. Results In contrast to M1 MΦ9s, both CD-derived M2 populations failed to develop the characteristic MΦ dendrite morphology seen in control macrophages. Surface expression of CD40 in M2 CD-derived MΦs was 3.4-fold lower for M2a and 4.4-fold lower for M2c compared to controls after E coli challenge. CD163 was higher in M1 CD cells but reduced by 50% in M2 cells compared to healthy cells. After E coli challenge, TNFα production by M2 but not M1 MΦs was significantly lower in CD than in controls (M2a 38%, M2c 27% respectively less than controls) but there were no differences in IL10 production. Prolonged intracellular survival of E coli was demonstrated in CD M2 cells but effective killing occurred in all M1 CD MΦs and all control MΦs. Conclusion In CD, M2 (but not M1) MΦs display abnormal morphological maturation, lower TNFα levels after E coli challenge, prolonged intracellular bacterial survival and differences in surface marker expression. The results are consistent with an innate MΦ defect in CD relating particularly to a failure of the normal role of M2 MΦs to limit and control inflammation.

STAT3-Mediated Production of Antimicrobial Peptides by Intestinal Epithelial Cells Helps Controlling C. rodentium Induced Infection

derived M2 populations failed to develop the characteristic MΦ dendrite morphology seen in control macrophages. Surface expression of CD40 in M2 CD-derived MΦs was 3.4-fold lower for M2a and 4.4fold lower for M2c compared to controls after E.coli challenge. CD163 was higher in M1 CD cells but reduced by 50% in M2 cells compared to healthy cells. After E.coli challenge, TNFα production by M2 but not M1 MΦs was significantly lower in CD than in controls (M2a 38%, M2c 27% respectively less than controls) but there were no differences in IL10 production. Prolonged intracellular survival of E.coli was demonstrated in CD M2 cells but effective killing occurred in all M1 CD MΦs and all control MΦs. In CD, M2 (but not M1) MΦs display abnormal morphological maturation, lower TNFα levels after E.coli challenge, prolonged intracellular bacterial survival and differences in surface marker expression. The results are consistent with an innate MΦ defect in CD relating particularly to a failure of the normal role of M2 MΦs to limit and control inflammation.

Defective macrophage function in crohn's disease: role of alternatively activated macrophages in inflammation

IntroductionThe aetiology of Crohn's disease (CD) involves a genetically determined dysregulated immune response to commensal intestinal microflora. In CD, viable E coli are found within lamina propria macrophages (MΦs) and...

Differences between peripheral blood and cord blood in the kinetics of lineage-restricted hematopoietic cells: implications for delayed platelet recovery following cord blood transplantation.

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However, the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity, i.e., CD34 and AC133. In addition to differences in surface marker expression, the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast, the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile, the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells.